Isolated human hepsin/46 proteins, nucleic acid molecules encoding human hepsin/46 proteins, and uses thereof

ABSTRACT

The present invention provides hepsin/46 protein, a soluble form of hepsin lacking the transmembrane region. The present invention specifically provides isolated peptide and nucleic acid molecules, methods of identifying molecules that interacting with the protease peptides, and methods of identifying modulators of the protease peptides.

FIELD OF THE INVENTION

[0001] The present invention is in the field of protease proteins thatare related to the hepsin, a serine protease subfamily, recombinant DNAmolecules, and protein production. The present invention specificallyprovides novel peptides and proteins that effect proteincleavage/processing/turnover and nucleic acid molecules encoding suchpeptide and protein molecules, all of which are useful in thedevelopment of human therapeutics and diagnostic compositions andmethods.

BACKGROUND OF THE INVENTION

[0002] The proteases may be categorized into families by the differentamino acid sequences (generally between 2 and 10 residues) located oneither side of the cleavage site of the protease.

[0003] The proper functioning of the cell requires careful control ofthe levels of important structural proteins, enzymes, and regulatoryproteins. One of the ways that cells can reduce the steady state levelof a particular protein is by proteolytic degradation. Further, one ofthe ways cells produce functioning proteins is to produce pre orpro-protein precursors that are processed by proteolytic degradation toproduce an active moiety. Thus, complex and highly-regulated mechanismshave been evolved to accomplish this degradation.

[0004] Proteases regulate many different cell proliferation,differentiation, and signaling processes by regulating protein turnoverand processing. Uncontrolled protease activity (either increased ordecreased) has been implicated in a variety of disease conditionsincluding inflammation, cancer, arteriosclerosis, and degenerativedisorders.

[0005] An additional role of intracellular proteolysis is in thestress-response. Cells that are subject to stress such as starvation,heat-shock, chemical insult or mutation respond by increasing the ratesof proteolysis. One function of this enhanced proteolysis is to salvageamino acids from non-essential proteins. These amino acids can then bere-utilized in the synthesis of essential proteins or metabolizeddirectly to provide energy. Another function is in the repair of damagecaused by the stress. For example, oxidative stress has been shown todamage a variety of proteins and cause them to be rapidly degraded.

[0006] The International Union of Biochemistry and Molecular Biology(IUBMB) has recommended to use the term peptidase for the subset ofpeptide bond hydrolases (Subclass E.C 3.4.). The widely used termprotease is synonymous with peptidase. Peptidases comprise two groups ofenzymes: the endopeptidases and the exopeptidases, which cleave peptidebonds at points within the protein and remove amino acids sequentiallyfrom either N or C-terminus respectively. The term proteinase is alsoused as a synonym word for endopeptidase and four mechanistic classes ofproteinases are recognized by the IUBMB: two of these are describedbelow (also see: Handbook of Proteolytic Enzymes by Barrett, Rawlings,and Woessner AP Press, NY 1998). Also, for a review of the various usesof proteases as drug targets, see: Weber M, Emerging treatments forhypertension: potential role for vasopeptidase inhibition; Am JHypertens 1999 November;12(11 Pt 2):139S-147S; Kentsch M, Otter W, Novelneurohormonal modulators in cardiovascular disorders. The therapeuticpotential of endopeptidase inhibitors, Drugs R D 1999 April;1(4):331-8;Scarborough R M, Coagulation factor Xa: the prothrombinase complex as anemerging therapeutic target for small molecule inhibitors, J Enzym Inhib1998;14(1):15-25; Skotnicki J S, et al., Design and syntheticconsiderations of matrix metalloproteinase inhibitors, Ann N Y Acad SciJun. 30, 1999;878:61-72; McKerrow J H, Engel J C, Caffrey C R, Cysteineprotease inhibitors as chemotherapy for parasitic infections, Bioorg MedChem 1999 April;7(4):639-44; Rice K D, Tanaka R D, Katz B A, Numerof RP, Moore W R, Inhibitors of tryptase for the treatment of mastcell-mediated diseases, Curr Pharm Des 1998 October;4(5):381-96;Materson B J, Will angiotensin converting enzyme genotype, receptormutation identification, and other miracles of molecular biology permitreduction of NNT. Am J Hypertens 1998 August;11(8 Pt 2):138S-142S.

[0007] Serine Proteases

[0008] The serine proteases (SP) are a large family of proteolyticenzymes that include the hepsin subfamily of proteins, the digestiveenzymes trypsin and chymotrypsin, components of the complement cascadeand of the blood-clotting cascade, and enzymes that control thedegradation and turnover of macromolecules of the extracellular matrix.SP are so named because of the presence of a serine residue in theactive catalytic site for protein cleavage. SP have a wide range ofsubstrate specificities and can be subdivided into subfamilies on thebasis of these specificities. The main sub-families are trypases(cleavage after arginine or lysine), aspases (cleavage after aspartate),chymases (cleavage after phenylalanine or leucine), metases (cleavageafter methionine), and serases (cleavage after serine).

[0009] A series of six SP have been identified in murine cytotoxicT-lymphocytes (CTL) and natural killer (NK) cells. These SP are involvedwith CTL and NK cells in the destruction of virally transformed cellsand tumor cells and in organ and tissue transplant rejection (Zunino, S.J. et al. (1990) J. Immunol. 144:2001-9; Sayers, T. J. et al. (1994) J.Immunol. 152:2289-97). Human homologs of most of these enzymes have beenidentified (Trapani, J. A. et al. (1988) Proc. Natl. Acad. Sci.85:6924-28; Caputo, A. et al. (1990) J. Immunol. 145:737-44). Like allSP, the CTL-SP share three distinguishing features: 1) the presence of acatalytic triad of histidine, serine, and aspartate residues whichcomprise the active site; 2) the sequence GDSGGP which contains theactive site serine; and 3) an N-terminal IIGG sequence whichcharacterizes the mature SP.

[0010] The SP are secretory proteins which contain N-terminal signalpeptides that serve to export the immature protein across theendoplasmic reticulum and are then cleaved (von Heijne (1986) Nuc. Acid.Res. 14:5683-90). Differences in these signal sequences provide onemeans of distinguishing individual SP. Some SP, particularly thedigestive enzymes, exist as inactive precursors or preproenzymes, andcontain a leader or activation peptide sequence 3′ of the signalpeptide. This activation peptide may be 2-12 amino acids in length, andit extends from the cleavage site of the signal peptide to theN-terminal IIGG sequence of the active, mature protein. Cleavage of thissequence activates the enzyme. This sequence varies in different SPaccording to the biochemical pathway and/or its substrate (Zunino et al,supra; Sayers et al, supra). Other features that distinguish various SPare the presence or absence of N-linked glycosylation sites that providemembrane anchors, the number and distribution of cysteine residues thatdetermine the secondary structure of the SP, and the sequence of asubstrate binding sites such as S′. The S′ substrate binding region isdefined by residues extending from approximately +17 to +29 relative tothe N-terminal I (+1). Differences in this region of the molecule arebelieved to determine SP substrate specificities (Zunino et al, supra).

[0011] The human hepsin cDNA was initially isolated from a liver cDNAlibrary screened with a mixture of oligonucleotides based on a consensussequence of serine proteases, Leytus et al., Biochemistry 27:1067-1074(1988). Biochemical studies indicate that hepsin is a type IItransmembrane serine protease expressed mainly on the surface ofhepatocytes, Tsugi et al., J. Biol. Chem. 266:16948-16953 (1991). Lowerlevels of hepsin mRNA are detected in other tissues including lung,kidney, pancreas, stomach, thyroid and prostate. In addition, hepsinmRNA is present in several human tumor cell lines, such as hepatomacells HepG2 and PLC/PRF/5, mammary cancer cells MCF784 and T470, andepitheloid carcinoma cells HeLa S3, Tsuji et al., J. Biol. Chem.266:16948-16953 (1991) and Torres-Rosado et al., Proc. Natl. Acad. Sci.USA 90:7181-7185 (1993).

[0012] Hepsin has a number of reported activities. In an in vitro study,recombinant human hepsin expressed on the cell surface activated bloodcoagulation factor VII but not factors IX, X, prothrombin or protein C,all of which share significant structural and sequence similarities withfactor VII. The activation of factor VII by hepsin was shown to besufficient to initiate the coagulation pathway leading to thrombinformation, Kazama et al., J. Biol. Chem. 270:66-72 (1995). Elevatedplasma factor VIIa activity has been known to be a significant riskfactor for ischemic heart disease and cardiovascular death, Hultin,Prog. Hemostasis Thrombosis 10:215-241, (1991) and Mann,Arteriosclerosis 9:783-784 (1989). Factor VIIa/tissue factor complexalso contributes to tumor-related hypercoagulability and intravascularthrombosis, Edwards et al., Thromb. Haemostasis 69:205-213 (1993).

[0013] In addition to blood coagulation, hepsin was reported to becritical for cell growth. In a cell culture system, addition ofanti-hepsin antibodies or hepsin-specific antisense oligonucleotides tothe culture medium significantly inhibited growth of hepatoma cells,Torres-Rosado et al., Proc. Natl. Acad. Sci. USA, 90:7181-7185 (1993).This observation is quite interesting in light of the expression ofhepsin mRNA in a number of tumor cells.

[0014] The growth factor-like activities of serine proteases have beenknown for many years. For example, thrombin is a potent mitogen forvascular fibroblasts and smooth muscle cells, Fenton J, Ann. N.Y. Acad.Sci., 485:5-15 (1986). Furthermore, serine proteases also participate inprocessing of growth factors, Massague J, J. Biol. Chem.,265:21393-21396 (1990). The hepsin-dependent tumor cell growth indicatesa mechanism in which hepsin functions either directly as a growth factoror indirectly as an enzyme that processes certain growth factorsessential for cell growth.

[0015] For a review of hepsin, see Tsuji et al., J. Biol. Chem.266:16948-16953, 1991; Tanimoto et al., Cancer Res. 57: 2884-2887, 1997;Zacharski, Thromb. Haemost. 79:876-877, 1998; Torres-Rosado et al.,Proc. Natl. Acad. Sci. 90:7181-7185, 1993; Kazama et al., J. Biol. Chem.270: 66-72, 1995; Luo et al., Cancer Res. 61: 4683-4688, 2001; Magee etal., Cancer Res. 61:5692-5696, 2001; Welsh, Cancer Res. 61: 5974-5978,2001; Dhanasekaran et al., Nature 412: 822-826, 2001; Stamey et al., J.Urol. 166:2171-2177, 2001; Vu et al., J. Biol. Chem. 272:31315-31320,1997.

[0016] Protease proteins, particularly hepsin, a member of serineprotease subfamily, are a major target for drug action and development.Accordingly, it is valuable to the field of pharmaceutical developmentto identify and characterize previously unknown members of thissubfamily of protease proteins. The present invention advances the stateof the art by providing a previously unidentified human hepsin protein.

SUMMARY OF THE INVENTION

[0017] The present invention provides a soluble form of human hepsin,hepsin/46. Hepsin/46 lacks the transmembrane region, starting at serine46. These unique peptide sequences, and nucleic acid sequences thatencode these peptides, can be used as models for the development ofhuman therapeutic targets, aid in the identification of therapeuticproteins, and serve as targets for the development of human therapeuticagents that modulate protease activity in cells and tissues that expressthe protease. The figure data provide evidence that a soluable form ofcatalytically active hepsin can be produced using the steps, orvariations of the steps, shown.

DESCRIPTION OF THE FIGURE SHEETS

[0018]FIG. 1 provides the nucleotide sequence of a cDNA molecule ortranscript sequence (SEQ ID NO: 1) that encodes the hepsin/46 protein ofthe present invention. (SEQ ID NO: 2). In addition the potentialtransmembrane domain is boxed, the protease activation site is indicatedby the solid triangle, and the catalytic triad in the serine proteasedomain is circled.

[0019]FIG. 2 provides the genetic sequence and the vector map of theplasmid used hepsin/46 sequence in the present invention. The pPIC9multiple cloning site and vector map provide a detailed analysis of thevarious points of interest of the vector.

[0020]FIG. 3 shows the purified hepsin 46 protein, after Sepharosepurification, through (A) a Zinc-stained gel and (B) a Western blot.

[0021]FIG. 4 shows the purified his-hepsin 46 protein, after Nickelpurification, through (A) a Silver-stained gel and (B) a Western blot.

[0022]FIG. 5 shows the purified recombinant active hepsin 46 protein,obtained after purification steps, by providing the agarose gelmigration of the heavy and light chain segment of hepsin 46.

DETAILED DESCRIPTION OF THE INVENTION

[0023] General Description

[0024] The present invention is based on the expression of truncatedform of human hepsin protein. Analysis of the sequence informationrevealed previously unidentified fragments of the hepsin peptides andits nucleotide sequence. Utilizing these sequences, additional genomicsequences were assembled and transcript and/or cDNA sequences wereisolated and characterized. Based on this analysis, the presentinvention provides amino acid sequences of human hepsin/46 proteins thatare related to the hepsin protease subfamily, nucleic acid sequences inthe form of transcript sequences, cDNA sequences and/or genomicsequences that encode these protease peptides and proteins, nucleic acidvariation (allelic information), tissue distribution of expression, andinformation about the closest art known protein/peptide/domain that hasstructural or sequence homology to the protease of the presentinvention.

[0025] In addition to being previously unknown, the peptides that areprovided in the present invention are selected based on their ability tobe used for the development of commercially important products andservices. Specifically, the present peptides are selected based ontruncating the hepsin protein and its binding activity to a substrate,and the expression pattern observed. The art has clearly established thecommercial importance of members of this family of proteins and proteinsthat have expression patterns similar to that of the present gene. Someof the more specific features of the peptides of the present invention,and the uses thereof, are described herein, particularly in theBackground of the Invention and in the data provided in the Figures.

[0026] Specific Embodiments

[0027] Peptide Molecules

[0028] The present invention provides nucleic acid sequences that encodeprotein molecules that have been identified as being members of theserine protease family of proteins and are related to the hepsin protein(protein sequences are provided in FIG. 1, cDNA sequences are providedin FIG. 1. The peptide sequences provided in FIG. 1, as well as theobvious variants described herein, will be referred herein as thehepsin/46 peptides of the present invention, hepsin protease peptides,or peptides/proteins of the present invention.

[0029] The present invention provides isolated peptide and proteinmolecules that consist of, consist essentially of, or comprise the aminoacid sequences of the hepsin/46 protease peptides disclosed in the FIG.1, (encoded by the nucleic acid molecule shown in FIG. 1, cDNA), as wellas all obvious variants of these peptides that are within the art tomake and use. Some of these variants are described in detail below.

[0030] As used herein, a peptide is said to be “isolated” or “purified”when it is substantially free of cellular material or free of chemicalprecursors or other chemicals. The peptides of the present invention canbe purified to homogeneity or other degrees of purity. The level ofpurification will be based on the intended use. The critical feature isthat the preparation allows for the desired function of the peptide,even if in the presence of considerable amounts of other components (thefeatures of an isolated nucleic acid molecule is discussed below).

[0031] In some uses, “substantially free of cellular material” includespreparations of the peptide having less than about 30% (by dry weight)other proteins (i.e., contaminating protein), less than about 20% otherproteins, less than about 10% other proteins, or less than about 5%other proteins. When the peptide is recombinantly produced, it can alsobe substantially free of culture medium, i.e., culture medium representsless than about 20% of the volume of the protein preparation.

[0032] The language “substantially free of chemical precursors or otherchemicals” includes preparations of the peptide in which it is separatedfrom chemical precursors or other chemicals that are involved in itssynthesis. In one embodiment, the language “substantially free ofchemical precursors or other chemicals” includes preparations of theprotease peptide having less than about 30% (by dry weight) chemicalprecursors or other chemicals, less than about 20% chemical precursorsor other chemicals, less than about 10% chemical precursors or otherchemicals, or less than about 5% chemical precursors or other chemicals.

[0033] The isolated protease peptide can be purified from cells thatnaturally express it, purified from cells that have been altered toexpress it (recombinant), or synthesized using known protein synthesismethods. For example, a nucleic acid molecule encoding the proteasepeptide is cloned into an expression vector, the expression vectorintroduced into a host cell and the protein expressed in the host cell.The protein can then be isolated from the cells by an appropriatepurification scheme using standard protein purification techniques. Manyof these techniques are described in detail below.

[0034] Accordingly, the present invention provides proteins that consistof the amino acid sequences provided in FIG. 1 (SEQ ID NO: 2), forexample, proteins encoded by the cDNA nucleic acid sequences shown inFIG. 1 (SEQ ID NO: 1). The amino acid sequence of such a protein isprovided in FIG. 1. A protein consists of an amino acid sequence whenthe amino acid sequence is the final amino acid sequence of the protein.

[0035] The present invention further provides proteins that consistessentially of the amino acid sequences provided in FIG. 1 (SEQ ID NO:2), for example, proteins encoded by the cDNA nucleic acid sequencesshown in FIG. 1 (SEQ ID NO:1) A protein consists essentially of an aminoacid sequence when such an amino acid sequence is present with only afew additional amino acid residues, for example from about 1 to about100 or so additional residues, typically from 1 to about 20 additionalresidues in the final protein. In one specific example, the protein wasadded additional six-histidine tags at its N terminal for purificationpurposes.

[0036] The present invention further provides proteins that comprise theamino acid sequences provided in FIG. 1 (SEQ ID NO: 2), for example,proteins encoded by the cDNA nucleic acid sequences shown in FIG. 1 (SEQID NO:1). A protein comprises an amino acid sequence when the amino acidsequence is at least part of the final amino acid sequence of theprotein. In such a fashion, the protein can be only the peptide or haveadditional amino acid molecules, such as amino acid residues (contiguousencoded sequence) that are naturally associated with it or heterologousamino acid residues/peptide sequences. Such a protein can have a fewadditional amino acid residues or can comprise several hundred or moreadditional amino acids. The preferred classes of proteins that arecomprised of the protease peptides of the present invention are thenaturally occurring mature proteins. A brief description of how varioustypes of these proteins can be made/isolated is provided below. In onespecific example, the protein was added additional six histidine tag atits N terminal for purification purposes.

[0037] The protease peptides of the present invention can be attached toheterologous sequences to form chimeric or fusion proteins. Suchchimeric and fusion proteins comprise a protease peptide operativelylinked to a heterologous protein having an amino acid sequence notsubstantially homologous to the protease peptide. “Operatively linked”indicates that the protease peptide and the heterologous protein arefused in-frame. The heterologous protein can be fused to the N-terminusor C-terminus of the protease peptide.

[0038] In some uses, the fusion protein does not affect the activity ofthe protease peptide per se. For example, the fusion protein caninclude, but is not limited to, enzymatic fusion proteins, for examplebeta-galactosidase fusions, yeast two-hybrid GAL fusions, poly-Hisfusions, MYC-tagged, HI-tagged and Ig fusions. Such fusion proteins,particularly poly-His fusions, can facilitate the purification ofrecombinant protease peptide. In certain host cells (e.g., mammalianhost cells), expression and/or secretion of a protein can be increasedby using a heterologous signal sequence.

[0039] A chimeric or fusion protein can be produced by standardrecombinant DNA techniques. For example, DNA fragments coding for thedifferent protein sequences are ligated together in-frame in accordancewith conventional techniques. In another embodiment, the fusion gene canbe synthesized by conventional techniques including automated DNAsynthesizers. Alternatively, PCR amplification of gene fragments can becarried out using anchor primers which give rise to complementaryoverhangs between two consecutive gene fragments which can subsequentlybe annealed and re-amplified to generate a chimeric gene sequence (seeAusubel et al., Current Protocols in Molecular Biology, 1992). Moreover,many expression vectors are commercially available that already encode afusion moiety (e.g., a GST protein). A protease peptide-encoding nucleicacid can be cloned into such an expression vector such that the fusionmoiety is linked in-frame to the protease peptide.

[0040] As mentioned above, the present invention also provides andenables obvious variants of the amino acid sequence of the proteins ofthe present invention, such as naturally occurring mature forms of thepeptide, allelic/sequence variants of the peptides, non-naturallyoccurring recombinantly derived variants of the peptides, and orthologsand paralogs of the peptides. Such variants can readily be generatedusing art-known techniques in the fields of recombinant nucleic acidtechnology and protein biochemistry. It is understood, however, thatvariants exclude any amino acid sequences disclosed prior to theinvention.

[0041] Such variants can readily be identified/made using moleculartechniques and the sequence information disclosed herein. Further, suchvariants can readily be distinguished from other peptides based onsequence and/or structural homology to the protease peptides of thepresent invention. The degree of homology/identity present will be basedprimarily on whether the peptide is a functional variant ornon-functional variant, the amount of divergence present in the paralogfamily and the evolutionary distance between the orthologs.

[0042] To determine the percent identity of two amino acid sequences ortwo nucleic acid sequences, the sequences are aligned for optimalcomparison purposes (e.g., gaps can be introduced in one or both of afirst and a second amino acid or nucleic acid sequence for optimalalignment and non-homologous sequences can be disregarded for comparisonpurposes). In a preferred embodiment, at least 30%, 40%, 50%, 60%, 70%,80%, 90%, 95%, 96%, 97%, 98% or 99% of the length of a referencesequence is aligned for comparison purposes. The amino acid residues ornucleotides at corresponding amino acid positions or nucleotidepositions are then compared. When a position in the first sequence isoccupied by the same amino acid residue or nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position (as used herein amino acid or nucleic acid“identity” is equivalent to amino acid or nucleic acid “homology”). Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences, taking into account thenumber of gaps, and the length of each gap, which need to be introducedfor optimal alignment of the two sequences.

[0043] The comparison of sequences and determination of percent identityand similarity between two sequences can be accomplished using amathematical algorithm. (Computational Molecular Biology, Lesk, A. M.,ed., Oxford University Press, New York, 1988; Biocomputing: Informaticsand Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993;Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and Griffin,H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis inMolecular Biology, von Heinje, G., Academic Press, 1987; and SequenceAnalysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press,New York, 1991). In a preferred embodiment, the percent identity betweentwo amino acid sequences is determined using the Needleman and Wunsch(J. Mol. Biol. (48):444-453 (1970)) algorithm which has beenincorporated into the GAP program in the GCG software package (availableat http://www.gcg.com), using either a Blossom 62 matrix or a PAM250matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a lengthweight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, thepercent identity between two nucleotide sequences is determined usingthe GAP program in the GCG software package (Devereux, J., et al.,Nucleic Acids Res. 12(1):387 (1984)) (available at http://www.gcg.com),using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80and a length weight of 1, 2, 3, 4, 5, or 6. In another embodiment, thepercent identity between two amino acid or nucleotide sequences isdetermined using the algorithm of E. Myers and W. Miller (CABIOS,4:11-17 (1989)) which has been incorporated into the ALIGN program(version 2.0), using a PAM120 weight residue table, a gap length penaltyof 12 and a gap penalty of 4.

[0044] The nucleic acid and protein sequences of the present inventioncan further be used as a “query sequence” to perform a search againstsequence databases to, for example, identify other family members orrelated sequences. Such searches can be performed using the NBLAST andXBLAST programs (version 2.0) of Altschul, et al. (J. Mol. Biol.215:403-10 (1990)). BLAST nucleotide searches can be performed with theNBLAST program, score=100, wordlength=12 to obtain nucleotide sequenceshomologous to the nucleic acid molecules of the invention. BLAST proteinsearches can be performed with the XBLAST program, score=50,wordlength=3 to obtain amino acid sequences homologous to the proteinsof the invention. To obtain gapped alignments for comparison purposes,Gapped BLAST can be utilized as described in Altschul et al. (NucleicAcids Res. 25(17):3389-3402 (1997)). When utilizing BLAST and gappedBLAST programs, the default parameters of the respective programs (e.g.,XBLAST and NBLAST) can be used.

[0045] Full-length pre-processed forms, as well as mature processedforms, of proteins that comprise one of the peptides of the presentinvention can readily be identified as having complete sequence identityto one of the protease peptides of the present invention as well asbeing encoded by the same genetic locus as the protease peptide providedherein. As indicated on FIG. 3, the hepsin protein gene sequence islocated on chromosome number 19 at q11-13.2.

[0046] Allelic variants of a protease peptide can readily be identifiedas being a human protein having a high degree (significant) of sequencehomology/identity to at least a portion of the protease peptide as wellas being encoded by the same genetic locus as the protease peptideprovided herein. As used herein, two proteins (or a region of theproteins) have significant homology when the amino acid sequences aretypically at least about −90%, and more typically at least about 90-95%or more homologous. A significantly homologous amino acid sequence,according to the present invention, will be encoded by a nucleic acidsequence that will hybridize to a protease peptide encoding nucleic acidmolecule under stringent conditions as more fully described below.

[0047] Paralogs of a protease peptide can readily be identified ashaving some degree of significant sequence homology/identity to at leasta portion of the protease peptide, as being encoded by a gene fromhumans, and as having similar activity or function. Two proteins willtypically be considered paralogs when the amino acid sequences aretypically at least about 60% or greater, and more typically at leastabout 70% or greater homology through a given region or domain. Suchparalogs will be encoded by a nucleic acid sequence that will hybridizeto a protease peptide encoding nucleic acid molecule under moderate tostringent conditions as more fully described below. In a preferredembodiment, the protein of the present invention comprises at least 278amino acids, wherein the amino acid is from position 93 to position 371of SEQ ID NO: 2.

[0048] Orthologs of a protease peptide can readily be identified ashaving some degree of significant sequence homology/identity to at leasta portion of the protease peptide as well as being encoded by a genefrom another organism. Preferred orthologs will be isolated frommammals, preferably primates, for the development of human therapeutictargets and agents. Such orthologs will be encoded by a nucleic acidsequence that will hybridize to a protease peptide encoding nucleic acidmolecule under moderate to stringent conditions, as more fully describedbelow, depending on the degree of relatedness of the two organismsyielding the proteins.

[0049] Non-naturally occurring variants of the protease peptides of thepresent invention can readily be generated using recombinant techniques.Such variants include, but are not limited to deletions, additions andsubstitutions in the amino acid sequence of the protease peptide. Forexample, one class of substitutions is conserved amino acidsubstitution. Such substitutions are those that substitute a given aminoacid in a protease peptide by another amino acid of likecharacteristics. Typically seen as conservative substitutions are thereplacements, one for another, among the aliphatic amino acids Ala, Val,Leu, and Ile; interchange of the hydroxyl residues Ser and Thr; exchangeof the acidic residues Asp and Glu; substitution between the amideresidues Asn and Gln; exchange of the basic residues Lys and Arg; andreplacements among the aromatic residues Phe and Tyr. Guidanceconcerning which amino acid changes are likely to be phenotypicallysilent are found in Bowie et al., Science 247:1306-1310 (1990). In apreferred embodiment, the protein of the present invention comprises atleast 278 amino acids, wherein the amino acid is from position 93 toposition 371 of SEQ ID NO: 2.

[0050] Variant protease peptides can be fully functional or can lackfunction in one or more activities, e.g. ability to bind substrate,ability to cleave substrate, ability to participate in a signalingpathway, etc. Fully functional variants typically contain onlyconservative variation or variation in non-critical residues or innon-critical regions. Functional variants can also contain substitutionof similar amino acids that result in no change or an insignificantchange in function. Alternatively, such substitutions may positively ornegatively affect function to some degree.

[0051] Non-functional variants typically contain one or morenon-conservative amino acid substitutions, deletions, insertions,inversions, or truncation or a substitution, insertion, inversion, ordeletion in a critical residue or critical region.

[0052] Amino acids that are essential for function can be identified bymethods known in the art, such as site-directed mutagenesis oralanine-scanning mutagenesis (Cunningham et al., Science 244:1081-1085(1989)). The latter procedure introduces single alanine mutations atevery residue in the molecule. The resulting mutant molecules are thentested for biological activity such as protease activity or in assayssuch as an in vitro proliferative activity. Sites that are critical forbinding partner/substrate binding can also be determined by structuralanalysis such as crystallization, nuclear magnetic resonance orphotoaffinity labeling (Smith et al., J Mol. Biol. 224:899-904 (1992);de Vos et al. Science 255:306-312 (1992)).

[0053] The present invention further provides fragments of the proteasepeptides, in addition to proteins and peptides that comprise and consistof such fragments. The fragments to which the invention pertains,however, are not to be construed as encompassing fragments that may bedisclosed publicly prior to the present invention.

[0054] As used herein, a fragment comprises at least 8, 10, 12, 14, 16,or more contiguous amino acid residues from a protease peptide. Suchfragments can be chosen based on the ability to retain one or more ofthe biological activities of the protease peptide or could be chosen forthe ability to perform a function, e.g. bind a substrate or act as animmunogen. Particularly important fragments are biologically activefragments, peptides that are, for example, about 8 or more amino acidsin length. Such fragments will typically comprise a domain or motif ofthe protease peptide, e.g., active site, a transmembrane domain or asubstrate-binding domain. Further, possible fragments include, but arenot limited to, domain or motif containing fragments, soluble peptidefragments, and fragments containing immunogenic structures.

[0055] Polypeptides often contain amino acids other than the 20 aminoacids commonly referred to as the 20 naturally occurring amino acids.Further, many amino acids, including the terminal amino acids, may bemodified by natural processes, such as processing and otherpost-translational modifications, or by chemical modification techniqueswell known in the art. Common modifications that occur naturally inprotease peptides are described in basic texts, detailed monographs, andthe research literature, and they are well known to those of skill inthe art.

[0056] Known modifications include, but are not limited to, acetylation,acylation, ADP-ribosylation, amidation, covalent attachment of flavin,covalent attachment of a heme moiety, covalent attachment of anucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of phosphotidylinositol,cross-linking, cyclization, disulfide bond formation, demethylation,formation of covalent crosslinks, formation of cystine, formation ofpyroglutamate, formylation, gamma carboxylation, glycosylation, GPIanchor formation, hydroxylation, iodination, methylation,myristoylation, oxidation, proteolytic processing, phosphorylation,prenylation, racemization, selenoylation, sulfation, transfer-RNAmediated addition of amino acids to proteins such as arginylation, andubiquitination.

[0057] Such modifications are well known to those of skill in the artand have been described in great detail in the scientific literature.Several particularly common modifications, glycosylation, lipidattachment, sulfation, gamma-carboxylation of glutamic acid residues,hydroxylation and ADP-ribosylation, for instance, are described in mostbasic texts, such as Proteins—Structure and Molecular Properties, 2ndEd., T. E. Creighton, W. H. Freeman and Company, New York (1993). Manydetailed reviews are available on this subject, such as by Wold, F.,Posttranslational Covalent Modification of Proteins, B. C. Johnson, Ed.,Academic Press, New York 1-12 (1983); Seifter et al. (Meth. Enzymol.182: 626-646 (1990)) and Rattan et al. (Ann. N.Y Acad. Sci. 663:48-62(1992)).

[0058] Accordingly, the protease peptides of the present invention alsoencompass derivatives or analogs in which a substituted amino acidresidue is not one encoded by the genetic code, in which a substitutedgroup is included, in which the mature protease peptide is fused withanother compound, such as a compound to increase the half-life of theprotease peptide (for example, polyethylene glycol), or in which theadditional amino acids are fused to the mature protease peptide, such asa leader or secretory sequence or a sequence for purification of themature protease peptide or a pro-protein sequence.

[0059] Protein/Peptide Uses

[0060] The proteins of the present invention can be used in substantialand specific assays related to the functional information provided inthe Figures; to raise antibodies or to elicit another immune response;as a reagent (including the labeled reagent) in assays designed toquantitatively determine levels of the protein (or its binding partneror ligand) in biological fluids; and as markers for tissues in which thecorresponding protein is preferentially expressed (either constitutivelyor at a particular stage of tissue differentiation or development or ina disease state). Where the protein binds or potentially binds toanother protein or ligand (such as, for example, in a protease-effectorprotein interaction or protease-ligand interaction), the protein can beused to identify the binding partner/ligand so as to develop a system toidentify inhibitors of the binding interaction. Any or all of these usesare capable of being developed into reagent grade or kit format forcommercialization as commercial products.

[0061] Methods for performing the uses listed above are well known tothose skilled in the art. References disclosing such methods include“Molecular Cloning: A Laboratory Manual”, 2d ed., Cold Spring HarborLaboratory Press, Sambrook, J., E. F. Fritsch and T. Maniatis eds.,1989, and “Methods in Enzymology: Guide to Molecular CloningTechniques”, Academic Press, Berger, S. L. and A. R. Kimmel eds., 1987.

[0062] The potential uses of the peptides of the present invention arebased primarily on the source of the protein as well as the class/actionof the protein. For example, proteases isolated from humans and theirhuman/mammalian orthologs serve as targets for identifying agents foruse in mammalian therapeutic applications, e.g. a human drug,particularly in modulating a biological or pathological response in acell or tissue that expresses the protease.

[0063] A large percentage of pharmaceutical agents are being developedthat modulate the activity of protease proteins, particularly members ofthe hepsin protease protein (see Background of the Invention). Thestructural and functional information provided in the Background andFigures provide specific and substantial uses for the molecules of thepresent invention. Such uses can readily be determined using theinformation provided herein, that which is known in the art, and routineexperimentation.

[0064] The proteins of the present invention (including variants andfragments that may have been disclosed prior to the present invention)are useful for biological assays related to proteases that are relatedto members of the hepsin protease protein. Such assays involve any ofthe known protease functions or activities or properties useful fordiagnosis and treatment of protease-related conditions that are specificfor the subfamily of proteases that the one of the present inventionbelongs to, particularly in cells and tissues that express the protease.

[0065] The proteins of the present invention are also useful in drugscreening assays, in cell-based or cell-free systems. Cell-based systemscan be native, i.e., cells that normally express the protease, as abiopsy or expanded in cell culture. In an alternate embodiment,cell-based assays involve recombinant host cells expressing the proteaseprotein.

[0066] The present invention provides a catalytically active humanhepsin protein. In particular, the invention provides methods of makinga truncated form of human hepsin that lacks the transmembrane region or,a form of human hepsin, which lacks the transmembrane region and anamino terminal His-tag.

[0067] The present invention provides a soluble form of human hepsin iscreated through a process which involved cloning the human hepsinsequence; starting at serine 46, express a truncated version with orwithout an amino terminal His-tag.

[0068] The present invention provides primers that are designed tointroduce an Xho I restriction site and regenerate the α-factor signalsequence. The DNA sequence encoding Hepsin/46; the expression constructwas generated and expressed in a histidine deficient yeast system.

[0069] The protein of present invention has specificity proteaseactivity using a chromagenic or fluorogenic substrate. The substratesinclude, but are not limited to the sequences comprising,Arg-Gln-Arg-Arg, Arg-Val-Arg-Arg, Pro-Arg, Gly-Arg-ArgAly-Gly-Pro-ArgAla-Arg, Gly-Gly-Arg, Gly-Pro-Arg, Glutaryl-Gly-Arg, Val-Pro-Arg, orGly-Pro-Arg, but preferably Arg-Gln-Arg-Arg.

[0070] The polypeptides can be used to identify compounds that modulateprotease activity of the protein in its natural state or an altered formthat causes a specific disease or pathology associated with theprotease. Both the proteases of the present invention and appropriatevariants and fragments can be used in high-throughput screens to assaycandidate compounds for the ability to bind to the protease. Thesecompounds can be further screened against a functional protease todetermine the effect of the compound on the protease activity. Further,these compounds can be tested in animal or invertebrate systems todetermine activity/effectiveness. Compounds can be identified thatactivate (agonist) or inactivate (antagonist) the protease to a desireddegree.

[0071] Further, the proteins of the present invention can be used toscreen a compound for the ability to stimulate or inhibit interactionbetween the protease protein and a molecule that normally interacts withthe protease protein, e.g. a substrate or a component of the signalpathway that the protease protein normally interacts (for example, aprotease). Such assays typically include the steps of combining theprotease protein with a candidate compound under conditions that allowthe protease protein, or fragment, to interact with the target molecule,and to detect the formation of a complex between the protein and thetarget or to detect the biochemical consequence of the interaction withthe protease protein and the target, such as any of the associatedeffects of signal transduction such as protein cleavage, cAMP turnover,and adenylate cyclase activation, etc.

[0072] Candidate compounds include, for example, 1) peptides such assoluble peptides, including Ig-tailed fusion peptides and members ofrandom peptide libraries (see, e.g., Lam et al., Nature 354:82-84(1991); Houghten et al., Nature 354:84-86 (1991)) and combinatorialchemistry-derived molecular libraries made of D- and/or L-configurationamino acids; 2) phosphopeptides (e.g., members of random and partiallydegenerate, directed phosphopeptide libraries, see, e.g., Songyang etal., Cell 72:767-778 (1993)); 3) antibodies (e.g., polyclonal,monoclonal, humanized, anti-idiotypic, chimeric, and single chainantibodies as well as Fab, F(ab′)₂, Fab expression library fragments,and epitope-binding fragments of antibodies); and 4) small organic andinorganic molecules (e.g., molecules obtained from combinatorial andnatural product libraries).

[0073] One candidate compound is a soluble fragment of the receptor thatcompetes for substrate binding. Other candidate compounds include mutantproteases or appropriate fragments containing mutations that affectprotease function and thus compete for substrate. Accordingly, afragment that competes for substrate, for example with a higheraffinity, or a fragment that binds substrate but does not allow release,is encompassed by the invention.

[0074] The invention further includes other end point assays to identifycompounds that modulate (stimulate or inhibit) protease activity. Theassays typically involve an assay of events in the signal transductionpathway that indicate protease activity. Thus, the cleavage of asubstrate, inactivation/activation of a protein, a change in theexpression of genes that are up- or down-regulated in response to theprotease protein dependent signal cascade can be assayed.

[0075] Any of the biological or biochemical functions mediated by theprotease can be used as an endpoint assay. These include all of thebiochemical or biochemical/biological events described herein, in thereferences cited herein, incorporated by reference for these endpointassay targets, and other functions known to those of ordinary skill inthe art or that can be readily identified using the information providedin the Figures. Specifically, a biological function of a cell or tissuesthat expresses the protease can be assayed. In one embodiment, theprotease activity of hepsin can be assayed using a chromagenic orfluorogenic substrate.

[0076] Binding and/or activating compounds can also be screened by usingchimeric protease proteins in which the amino terminal extracellulardomain, or parts thereof, the entire transmembrane domain or subregions,such as any of the seven transmembrane segments or any of theintracellular or extracellular loops and the carboxyl terminalintracellular domain, or parts thereof, can be replaced by heterologousdomains or subregions. For example, a substrate-binding region can beused that interacts with a different substrate then that which isrecognized by the native protease. Accordingly, a different set ofsignal transduction components is available as an end-point assay foractivation. This allows for assays to be performed in other than thespecific host cell from which the protease is derived.

[0077] The proteins of the present invention are also useful incompetition binding assays in methods designed to discover compoundsthat interact with the protease (e.g. binding partners and/or ligands).Thus, a compound is exposed to a protease polypeptide under conditionsthat allow the compound to bind or to otherwise interact with thepolypeptide. Soluble protease polypeptide is also added to the mixture.If the test compound interacts with the soluble protease polypeptide, itdecreases the amount of complex formed or activity from the proteasetarget. This type of assay is particularly useful in cases in whichcompounds are sought that interact with specific regions of theprotease. Thus, the soluble polypeptide that competes with the targetprotease region is designed to contain peptide sequences correspondingto the region of interest.

[0078] To perform cell free drug screening assays, it is sometimesdesirable to immobilize either the protease protein, or fragment, or itstarget molecule to facilitate separation of complexes from uncomplexedforms of one or both of the proteins, as well as to accommodateautomation of the assay.

[0079] Techniques for immobilizing proteins on matrices can be used inthe drug screening assays. In one embodiment, a fusion protein can beprovided which adds a domain that allows the protein to be bound to amatrix. For example, glutathione-S-transferase fusion proteins can beadsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis,Mo.) or glutathione derivatized microtitre plates, which are thencombined with the cell lysates (e.g., ³⁵S-labeled) and the candidatecompound, and the mixture incubated under conditions conducive tocomplex formation (e.g., at physiological conditions for salt and pH).Following incubation, the beads are washed to remove any unbound label,and the matrix immobilized and radiolabel determined directly, or in thesupernatant after the complexes are dissociated. Alternatively, thecomplexes can be dissociated from the matrix, separated by SDS-PAGE, andthe level of protease-binding protein found in the bead fractionquantitated from the gel using standard electrophoretic techniques. Forexample, either the polypeptide or its target molecule can beimmobilized utilizing conjugation of biotin and streptavidin usingtechniques well known in the art. Alternatively, antibodies reactivewith the protein but which do not interfere with binding of the proteinto its target molecule can be derivatized to the wells of the plate, andthe protein trapped in the wells by antibody conjugation. Preparationsof a protease-binding protein and a candidate compound are incubated inthe protease protein-presenting wells and the amount of complex trappedin the well can be quantitated. Methods for detecting such complexes, inaddition to those described above for the GST-immobilized complexes,include immunodetection of complexes using antibodies reactive with theprotease protein target molecule, or which are reactive with proteaseprotein and compete with the target molecule, as well as enzyme-linkedassays which rely on detecting an enzymatic activity associated with thetarget molecule.

[0080] Agents that modulate one of the proteases of the presentinvention can be identified using one or more of the above assays, aloneor in combination. It is generally preferable to use a cell-based orcell free system first and then confirm activity in an animal or othermodel system. Such model systems are well known in the art and canreadily be employed in this context.

[0081] Modulators of protease protein activity identified according tothese drug screening assays can be used to treat a subject with adisorder mediated by the protease pathway, by treating cells or tissuesthat express the protease. These methods of treatment include the stepsof administering a modulator of protease activity in a pharmaceuticalcomposition to a subject in need of such treatment, the modulator beingidentified as described herein.

[0082] In yet another aspect of the invention, the protease proteins canbe used as “bait proteins” in a two-hybrid assay or three-hybrid assay(see, e.g., U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell72:223-232; Madura et al. (1993) J. Biol. Chem. 268:12046-12054; Bartelet al. (1993) Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene8:1693-1696; and Brent WO94/10300), to identify other proteins, whichbind to or interact with the protease and are involved in proteaseactivity. Such protease-binding proteins are also likely to be involvedin the propagation of signals by the protease proteins or proteasetargets as, for example, downstream elements of a protease-mediatedsignaling pathway. Alternatively, such protease-binding proteins arelikely to be protease inhibitors.

[0083] The two-hybrid system is based on the modular nature of mosttranscription factors, which consist of separable DNA-binding andactivation domains. Briefly, the assay utilizes two different DNAconstructs. In one construct, the gene that codes for a protease proteinis fused to a gene encoding the DNA binding domain of a knowntranscription factor (e.g., GAL-4). In the other construct, a DNAsequence, from a library of DNA sequences, that encodes an unidentifiedprotein (“prey” or “sample”) is fused to a gene that codes for theactivation domain of the known transcription factor. If the “bait” andthe “prey” proteins are able to interact, in vivo, forming aprotease-dependent complex, the DNA-binding and activation domains ofthe transcription factor are brought into close proximity. Thisproximity allows transcription of a reporter gene (e.g., LacZ) which isoperably linked to a transcriptional regulatory site responsive to thetranscription factor. Expression of the reporter gene can be detectedand cell colonies containing the functional transcription factor can beisolated and used to obtain the cloned gene which encodes the proteinwhich interacts with the protease protein.

[0084] This invention further pertains to novel agents identified by theabove-described screening assays. Accordingly, it is within the scope ofthis invention to further use an agent identified as described herein inan appropriate animal model. For example, an agent identified asdescribed herein (e.g., a protease-modulating agent, an antisenseprotease nucleic acid molecule, a protease-specific antibody, or aprotease-binding partner) can be used in an animal or other model todetermine the efficacy, toxicity, or side effects of treatment with suchan agent. Alternatively, an agent identified as described herein can beused in an animal or other model to determine the mechanism of action ofsuch an agent. Furthermore, this invention pertains to uses of novelagents identified by the above-described screening assays for treatmentsas described herein.

[0085] The protease proteins of the present invention are also useful toprovide a target for diagnosing a disease or predisposition to diseasemediated by the peptide. Accordingly, the invention provides methods fordetecting the presence, or levels of, the protein (or encoding mRNA) ina cell, tissue, or organism. The method involves contacting a biologicalsample with a compound capable of interacting with the protease proteinsuch that the interaction can be detected. Such an assay can be providedin a single detection format or a multi-detection format such as anantibody chip array.

[0086] One agent for detecting a protein in a sample is an antibodycapable of selectively binding to protein. A biological sample includestissues, cells and biological fluids isolated from a subject, as well astissues, cells and fluids present within a subject.

[0087] The peptides of the present invention also provide targets fordiagnosing active protein activity, disease, or predisposition todisease, in a patient having a variant peptide, particularly activitiesand conditions that are known for other members of the family ofproteins to which the present one belongs. Thus, the peptide can beisolated from a biological sample and assayed for the presence of agenetic mutation that results in aberrant peptide. This includes aminoacid substitution, deletion, insertion, rearrangement, (as the result ofaberrant splicing events), and inappropriate post-translationalmodification. Analytic methods include altered electrophoretic mobility,altered tryptic peptide digest, altered protease activity in cell-basedor cell-free assay, alteration in substrate or antibody-binding pattern,altered isoelectric point, direct amino acid sequencing, and any otherof the known assay techniques useful for detecting mutations in aprotein. Such an assay can be provided in a single detection format or amulti-detection format such as an antibody chip array.

[0088] In vitro techniques for detection of peptide include enzymelinked immunosorbent assays (ELISAs), Western blots,immunoprecipitations and immunofluorescence using a detection reagent,such as an antibody or protein binding agent. Alternatively, the peptidecan be detected in vivo in a subject by introducing into the subject alabeled anti-peptide antibody or other types of detection agent. Forexample, the antibody can be labeled with a radioactive marker whosepresence and location in a subject can be detected by standard imagingtechniques. Particularly useful are methods that detect the allelicvariant of a peptide expressed in a subject and methods which detectfragments of a peptide in a sample.

[0089] The peptides are also useful in pharmacogenomic analysis.Pharmacogenomics deal with clinically significant hereditary variationsin the response to drugs due to altered drug disposition and abnormalaction in affected persons. See, e.g., Eichelbaum, M. (Clin. Exp.Pharmacol. Physiol. 23(10-11):983-985 (1996)), and Linder, M. W. (Clin.Chem. 43(2):254-266 (1997)). The clinical outcomes of these variationsresult in severe toxicity of therapeutic drugs in certain individuals ortherapeutic failure of drugs in certain individuals as a result ofindividual variation in metabolism. Thus, the genotype of the individualcan determine the way a therapeutic compound acts on the body or the waythe body metabolizes the compound. Further, the activity of drugmetabolizing enzymes effects both the intensity and duration of drugaction. Thus, the pharmacogenomics of the individual permit theselection of effective compounds and effective dosages of such compoundsfor prophylactic or therapeutic treatment based on the individual'sgenotype. The discovery of genetic polymorphisms in some drugmetabolizing enzymes has explained why some patients do not obtain theexpected drug effects, show an exaggerated drug effect, or experienceserious toxicity from standard drug dosages. Polymorphisms can beexpressed in the phenotype of the extensive metabolizer and thephenotype of the poor metabolizer. Accordingly, genetic polymorphism maylead to allelic protein variants of the protease protein in which one ormore of the protease functions in one population is different from thosein another population. The peptides thus allow a target to ascertain agenetic predisposition that can affect treatment modality. Thus, in aligand-based treatment, polymorphism may give rise to amino terminalextracellular domains and/or other substrate-binding regions that aremore or less active in substrate binding, and protease activation.Accordingly, substrate dosage would necessarily be modified to maximizethe therapeutic effect within a given population containing apolymorphism. As an alternative to genotyping, specific polymorphicpeptides could be identified.

[0090] The peptides are also useful for treating a disordercharacterized by an absence of, inappropriate, or unwanted expression ofthe protein. Accordingly, methods for treatment include the use of theprotease protein or fragments.

[0091] Antibodies

[0092] The invention also provides antibodies that selectively bind toone of the peptides of the present invention, a protein comprising sucha peptide, as well as variants and fragments thereof. As used herein, anantibody selectively binds a target peptide when it binds the targetpeptide and does not significantly bind to unrelated proteins. Anantibody is still considered to selectively bind a peptide even if italso binds to other proteins that are not substantially homologous withthe target peptide so long as such proteins share homology with afragment or domain of the peptide target of the antibody. In this case,it would be understood that antibody binding to the peptide is stillselective despite some degree of cross-reactivity.

[0093] As used herein, an antibody is defined in terms consistent withthat recognized within the art: they are multi-subunit proteins producedby a mammalian organism in response to an antigen challenge. Theantibodies of the present invention include polyclonal antibodies andmonoclonal antibodies, as well as fragments of such antibodies,including, but not limited to, Fab or F(ab′)₂, and Fv fragments.

[0094] Many methods are known for generating and/or identifyingantibodies to a given target peptide. Several such methods are describedby Harlow, Antibodies, Cold Spring Harbor Press, (1989).

[0095] In general, to generate antibodies, an isolated peptide is usedas an immunogen and is administered to a mammalian organism, such as arat, rabbit or mouse. The full-length protein, an antigenic peptidefragment or a fusion protein can be used. Antibodies are preferablyprepared from regions or discrete fragments of the protease proteins.Antibodies can be prepared from any region of the peptide as describedherein. However, preferred regions will include those involved infunction/activity and/or protease/binding partner interaction.

[0096] An antigenic fragment will typically comprise at least 8contiguous amino acid residues. The antigenic peptide can comprise,however, at least 10, 12, 14, 16 or more amino acid residues. Suchfragments can be selected on a physical property, such as fragmentscorrespond to regions that are located on the surface of the protein.

[0097] Detection on an antibody of the present invention can befacilitated by coupling (i.e., physically linking) the antibody to adetectable substance. Examples of detectable substances include variousenzymes, prosthetic groups, fluorescent materials, luminescentmaterials, bioluminescent materials, and radioactive materials. Examplesof suitable enzymes include horseradish peroxidase, alkalinephosphatase, β-galactosidase, or acetylcholinesterase; examples ofsuitable prosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; examples ofbioluminescent materials include luciferase, luciferin, and aequorin,and examples of suitable radioactive material include ¹²⁵I, ¹³¹I, ³⁵S or³H.

[0098] Antibody Uses

[0099] The antibodies can be used to isolate one of the proteins of thepresent invention by standard techniques, such as affinitychromatography or immunoprecipitation. The antibodies can facilitate thepurification of the natural protein from cells and recombinantlyproduced protein expressed in host cells. In addition, such antibodiesare useful to detect the presence of one of the proteins of the presentinvention in cells or tissues to determine the pattern of expression ofthe protein among various tissues in an organism and over the course ofnormal development. Further, such antibodies can be used to detectprotein in situ, in vitro, or in a cell lysate or supernatant in orderto evaluate the abundance and pattern of expression. Also, suchantibodies can be used to assess abnormal tissue distribution orabnormal expression during development or progression of a biologicalcondition. Antibody detection of circulating fragments of thefull-length protein can be used to identify turnover.

[0100] Further, the antibodies can be used to assess expression indisease states such as in active stages of the disease or in anindividual with a predisposition toward disease related to the protein'sfunction. When a disorder is caused by an inappropriate tissuedistribution, developmental expression, level of expression of theprotein, or expressed/processed form, the antibody can be preparedagainst the normal protein. If a disorder is characterized by a specificmutation in the protein, antibodies specific for this mutant protein canbe used to assay for the presence of the specific mutant protein.

[0101] The antibodies can also be used to assess normal and aberrantsubcellular localization of cells in the various tissues in an organism.The diagnostic uses can be applied, not only in genetic testing, butalso in monitoring a treatment modality. Accordingly, where treatment isultimately aimed at correcting expression level or the presence ofaberrant sequence and aberrant tissue distribution or developmentalexpression, antibodies directed against the protein or relevantfragments can be used to monitor therapeutic efficacy.

[0102] Additionally, antibodies are useful in pharmacogenomic analysis.Thus, antibodies prepared against polymorphic proteins can be used toidentify individuals that require modified treatment modalities. Theantibodies are also useful as diagnostic tools as an immunologicalmarker for aberrant protein analyzed by electrophoretic mobility,isoelectric point, tryptic peptide digest, and other physical assaysknown to those in the art.

[0103] The antibodies are also useful for tissue typing. Levels ofhepsin mRNA are detected in other tissues including lung, kidney,pancreas, stomach, thyroid and prostate. Thus, where a specific proteinhas been correlated with expression in a specific tissue, antibodiesthat are specific for this protein can be used to identify a tissuetype.

[0104] The antibodies are also useful for inhibiting protein function,for example, blocking the binding of the protease peptide to a bindingpartner such as a substrate. These uses can also be applied in atherapeutic context in which treatment involves inhibiting the protein'sfunction. An antibody can be used, for example, to block binding, thusmodulating (agonizing or antagonizing) the peptides activity. Antibodiescan be prepared against specific fragments containing sites required forfunction or against intact protein that is associated with a cell orcell membrane.

[0105] The invention also encompasses kits for using antibodies todetect the presence of a protein in a biological sample. The kit cancomprise antibodies such as a labeled or labelable antibody and acompound or agent for detecting protein in a biological sample; meansfor determining the amount of protein in the sample; means for comparingthe amount of protein in the sample with a standard; and instructionsfor use. Such a kit can be supplied to detect a single protein orepitope or can be configured to detect one of a multitude of epitopes,such as in an antibody detection array. Arrays are described in detailbelow for nucleic acid arrays and similar methods have been developedfor antibody arrays.

[0106] Nucleic Acid Molecules

[0107] The present invention further provides isolated nucleic acidmolecules that encode a protease peptide or protein of the presentinvention (cDNA, transcript and genomic sequence). Such nucleic acidmolecules will consist of, consist essentially of, or comprise anucleotide sequence that encodes one of the protease peptides of thepresent invention, an allelic variant thereof, or an ortholog or paralogthereof.

[0108] As used herein, an “isolated” nucleic acid molecule is one thatis separated from other nucleic acid present in the natural source ofthe nucleic acid. Preferably, an “isolated” nucleic acid is free ofsequences, which naturally flank the nucleic acid (i.e., sequenceslocated at the 5′ and 3′ ends of the nucleic acid) in the genomic DNA ofthe organism from which the nucleic acid is derived. However, there canbe some flanking nucleotide sequences, for example up to about 5 KB, 4KB, 3 KB, 2 KB, or 1 KB or less, particularly contiguous peptideencoding sequences and peptide encoding sequences within the same genebut separated by introns in the genomic sequence. The important point isthat the nucleic acid is isolated from remote and unimportant flankingsequences such that it can be subjected to the specific manipulationsdescribed herein such as recombinant expression, preparation of probesand primers, and other uses specific to the nucleic acid sequences.

[0109] Moreover, an “isolated” nucleic acid molecule, such as a cDNAmolecule, can be substantially free of other cellular material, orculture medium when produced by recombinant techniques, or chemicalprecursors or other chemicals when chemically synthesized. However, thenucleic acid molecule can be fused to other coding or regulatorysequences and still be considered isolated.

[0110] For example, recombinant DNA molecules contained in a vector areconsidered isolated. Further examples of isolated DNA molecules includerecombinant DNA molecules maintained in heterologous host cells orpurified (partially or substantially) DNA molecules in solution.Isolated RNA molecules include in vivo or in vitro RNA transcripts ofthe isolated DNA molecules of the present invention. Isolated nucleicacid molecules according to the present invention further include suchmolecules produced synthetically.

[0111] Accordingly, the present invention provides nucleic acidmolecules that consist of the nucleotide sequence shown in FIG. 1 (SEQID NO:1, transcript sequence), or any nucleic acid molecule that encodesthe protein provided in FIG. 1, SEQ ID NO:2. A nucleic acid moleculeconsists of a nucleotide sequence when the nucleotide sequence is thecomplete nucleotide sequence of the nucleic acid molecule.

[0112] The present invention further provides nucleic acid moleculesthat consist essentially of the nucleotide sequence shown in FIG. 1 (SEQID NO: 1, transcript sequence), or any nucleic acid molecule thatencodes the protein provided in FIG. 1, SEQ ID NO:2. A nucleic acidmolecule consists essentially of a nucleotide sequence when such anucleotide sequence is present with only a few additional nucleic acidresidues in the final nucleic acid molecule.

[0113] The present invention further provides nucleic acid moleculesthat comprise the nucleotide sequences shown in FIG. 1 (SEQ ID NO: 1,transcript sequence), or any nucleic acid molecule that encodes theprotein provided in FIG. 1, SEQ ID NO: 2. A nucleic acid moleculecomprises a nucleotide sequence when the nucleotide sequence is at leastpart of the final nucleotide sequence of the nucleic acid molecule. Insuch a fashion, the nucleic acid molecule can be only the nucleotidesequence or have additional nucleic acid residues, such as nucleic acidresidues that are naturally associated with it or heterologousnucleotide sequences. Such a nucleic acid molecule can have a fewadditional nucleotides or can comprise several hundred or moreadditional nucleotides. A brief description of how various types ofthese nucleic acid molecules can be readily made/isolated is providedbelow.

[0114] The isolated nucleic acid molecules can encode the mature proteinplus additional amino or carboxyl-terminal amino acids, or amino acidsinterior to the mature peptide (when the mature form has more than onepeptide chain, for instance). Such sequences may play a role inprocessing of a protein from precursor to a mature form, facilitateprotein trafficking, prolong or shorten protein half-life or facilitatemanipulation of a protein for assay or production, among other things.As generally is the case in situ, the additional amino acids may beprocessed away from the mature protein by cellular enzymes.

[0115] Isolated nucleic acid molecules can be in the form of RNA, suchas mRNA, or in the form DNA, including cDNA and genomic DNA obtained bycloning or produced by chemical synthetic techniques or by a combinationthereof. The nucleic acid, especially DNA, can be double-stranded orsingle-stranded. Single-stranded nucleic acid can be the coding strand(sense strand) or the non-coding strand (anti-sense strand).

[0116] The invention further provides nucleic acid molecules that encodefragments of the peptides of the present invention as well as nucleicacid molecules that encode obvious variants of the protease proteins ofthe present invention that are described above. Such nucleic acidmolecules may be naturally occurring, such as allelic variants (samelocus), paralogs (different locus), and orthologs (different organism),or may be constructed by recombinant DNA methods or by chemicalsynthesis. Such non-naturally occurring variants may be made bymutagenesis techniques, including those applied to nucleic acidmolecules, cells, or organisms. Accordingly, as discussed above, thevariants can contain nucleotide substitutions, deletions, inversions andinsertions. Variation can occur in either or both the coding andnon-coding regions. The variations can produce both conservative andnon-conservative amino acid substitutions.

[0117] A fragment comprises a contiguous nucleotide sequence greaterthan 12 or more nucleotides. Further, a fragment could at least 30, 40,50, 100, 250 or 500 nucleotides in length. The length of the fragmentwill be based on its intended use. For example, the fragment can encodeepitope bearing regions of the peptide, or can be useful as DNA probesand primers. Such fragments can be isolated using the known nucleotidesequence to synthesize an oligonucleotide probe. A labeled probe canthen be used to screen a cDNA library, genomic DNA library, or mRNA toisolate nucleic acid corresponding to the coding region. Further,primers can be used in PCR reactions to clone specific regions of gene.

[0118] A probe/primer typically comprises substantially a purifiedoligonucleotide or oligonucleotide pair. The oligonucleotide typicallycomprises a region of nucleotide sequence that hybridizes understringent conditions to at least about 12, 20, 25, 40, 50 or moreconsecutive nucleotides.

[0119] Orthologs, homologs, and allelic variants can be identified usingmethods well known in the art. As described in the Peptide Section,these variants comprise a nucleotide sequence encoding a peptide that istypically 60-70%, 70-80%, 80-90%, and more typically at least about90-95% or more homologous to the nucleotide sequence shown in the Figuresheets or a fragment of this sequence. Such nucleic acid molecules canreadily be identified as being able to hybridize under moderate tostringent conditions, to the nucleotide sequence shown in the Figuresheets or a fragment of the sequence. Allelic variants can readily bedetermined by genetic locus of the encoding gene.

[0120] As used herein, the term “hybridizes under stringent conditions”is intended to describe conditions for hybridization and washing underwhich nucleotide sequences encoding a peptide at least 60-70% homologousto each other typically remain hybridized to each other. The conditionscan be such that sequences at least about 60%, at least about 70%, or atleast about 80% or more homologous to each other typically remainhybridized to each other. Such stringent conditions are known to thoseskilled in the art and can be found in Current Protocols in MolecularBiology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. One example ofstringent hybridization conditions are hybridization in 6× sodiumchloride/sodium citrate (SSC) at about 45C, followed by one or morewashes in 0.2×SSC, 0.1% SDS at 50-65C. Examples of moderate to lowstringency hybridization conditions are well known in the art.

[0121] Nucleic Acid Molecule Uses

[0122] The nucleic acid molecules of the present invention are usefulfor probes, primers, chemical intermediates, and in biological assays.The nucleic acid molecules are useful as a hybridization probe formessenger RNA, cDNA to isolate full-length cDNA and genomic clonesencoding the peptide described in FIG. 1 and to isolate cDNA and genomicclones that correspond to variants (alleles, orthologs, etc.) producingthe same or related peptides shown in FIG. 1. SEQ ID NO: 2 representsthe truncated form of active human hepsin which allows for a solubleversion of the normally transmembrane bound protein.

[0123] The probe can correspond to any sequence along the entire lengthof the nucleic acid molecules provided in the Figures. Accordingly, itcould be derived from any part of the coding region. However, asdiscussed, fragments are not to be construed as encompassing fragmentsdisclosed prior to the present invention.

[0124] The nucleic acid molecules are also useful as primers for PCR toamplify any given region of a nucleic acid molecule and are useful tosynthesize antisense molecules of desired length and sequence.

[0125] The nucleic acid molecules are also useful for constructingrecombinant vectors. Such vectors include expression vectors thatexpress a portion of, or all of, the peptide sequences. Vectors alsoinclude insertion vectors, used to integrate into another nucleic acidmolecule sequence, such as into the cellular genome, to alter in situexpression of a gene and/or gene product. For example, an endogenouscoding sequence can be replaced via homologous recombination with all orpart of the coding region containing one or more specifically introducedmutations.

[0126] The nucleic acid molecules are also useful for expressingantigenic portions of the proteins.

[0127] The nucleic acid molecules are also useful as probes fordetermining the chromosomal positions of the nucleic acid molecules bymeans of in situ hybridization methods.

[0128] The nucleic acid molecules are also useful in making vectorscontaining the gene regulatory regions of the nucleic acid molecules ofthe present invention.

[0129] The nucleic acid molecules are also useful for designingribozymes corresponding to all, or a part, of the mRNA produced from thenucleic acid molecules described herein.

[0130] The nucleic acid molecules are also useful for making vectorsthat express part, or all, of the peptides.

[0131] The nucleic acid molecules are also useful for constructing hostcells expressing a part, or all, of the nucleic acid molecules andpeptides.

[0132] The nucleic acid molecules are also useful for constructingtransgenic animals expressing all, or a part, of the nucleic acidmolecules and peptides.

[0133] The nucleic acid molecules are also useful as hybridizationprobes for determining the presence, level, form and distribution ofnucleic acid expression. Accordingly, the probes can be used to detectthe presence of, or to determine levels of, a specific nucleic acidmolecule in cells, tissues, and in organisms. The nucleic acid whoselevel is determined can be DNA or RNA. Accordingly, probes correspondingto the peptides described herein can be used to assess expression and/orgene copy number in a given cell, tissue, or organism. These uses arerelevant for diagnosis of disorders involving an increase or decrease inprotease protein expression relative to normal results.

[0134] In vitro techniques for detection of mRNA include Northernhybridizations and in situ hybridizations. In vitro techniques fordetecting DNA includes Southern hybridizations and in situhybridization.

[0135] Probes can be used as a part of a diagnostic test kit foridentifying cells or tissues that express a protease protein, such as bymeasuring a level of a protease-encoding nucleic acid in a sample ofcells from a subject e.g., mRNA or genomic DNA, or determining if aprotease gene has been mutated.

[0136] Nucleic acid expression assays are useful for drug screening toidentify compounds that modulate protease nucleic acid expression.

[0137] The invention thus provides a method for identifying a compoundthat can be used to treat a disorder associated with nucleic acidexpression of the protease gene, particularly biological andpathological processes that are mediated by the protease in cells andtissues that express it. The method typically includes assaying theability of the compound to modulate the expression of the proteasenucleic acid and thus identifying a compound that can be used to treat adisorder characterized by undesired protease nucleic acid expression.The assays can be performed in cell-based and cell-free systems.Cell-based assays include cells naturally expressing the proteasenucleic acid or recombinant cells genetically engineered to expressspecific nucleic acid sequences.

[0138] The assay for protease nucleic acid expression can involve directassay of nucleic acid levels, such as mRNA levels, or on collateralcompounds involved in the signal pathway. Further, the expression ofgenes that are up- or down-regulated in response to the protease proteinsignal pathway can also be assayed. In this embodiment the regulatoryregions of these genes can be operably linked to a reporter gene such asluciferase.

[0139] Thus, modulators of protease gene expression can be identified ina method wherein a cell is contacted with a candidate compound and theexpression of mRNA determined. The level of expression of protease mRNAin the presence of the candidate compound is compared to the level ofexpression of protease mRNA in the absence of the candidate compound.The candidate compound can then be identified as a modulator of nucleicacid expression based on this comparison and be used, for example totreat a disorder characterized by aberrant nucleic acid expression. Whenexpression of mRNA is statistically significantly greater in thepresence of the candidate compound than in its absence, the candidatecompound is identified as a stimulator of nucleic acid expression. Whennucleic acid expression is statistically significantly less in thepresence of the candidate compound than in its absence, the candidatecompound is identified as an inhibitor of nucleic acid expression.

[0140] The invention further provides methods of treatment, with thenucleic acid as a target, using a compound identified through drugscreening as a gene modulator to modulate protease nucleic acidexpression in cells and tissues that express the protease. Modulationincludes both up-regulation (i.e. activation or agonization) ordown-regulation (suppression or antagonization) or nucleic acidexpression.

[0141] Alternatively, a modulator for protease nucleic acid expressioncan be a small molecule or drug identified using the screening assaysdescribed herein as long as the drug or small molecule inhibits theprotease nucleic acid expression in the cells and tissues that expressthe protein. For the present invention, the expressive tissues includelung, kidney, liver and prostate and ovarian cancer cell lines.

[0142] The nucleic acid molecules are also useful for monitoring theeffectiveness of modulating compounds on the expression or activity ofthe protease gene in clinical trials or in a treatment regimen. Thus,the gene expression pattern can serve as a barometer for the continuingeffectiveness of treatment with the compound, particularly withcompounds to which a patient can develop resistance. The gene expressionpattern can also serve as a marker indicative of a physiologicalresponse of the affected cells to the compound. Accordingly, suchmonitoring would allow either increased administration of the compoundor the administration of alternative compounds to which the patient hasnot become resistant. Similarly, if the level of nucleic acid expressionfalls below a desirable level, administration of the compound could becommensurately decreased.

[0143] The nucleic acid molecules are also useful in diagnostic assaysfor qualitative changes in protease nucleic acid expression, andparticularly in qualitative changes that lead to pathology. The nucleicacid molecules can be used to detect mutations in protease genes andgene expression products such as mRNA. The nucleic acid molecules can beused as hybridization probes to detect naturally occurring geneticmutations in the protease gene and thereby to determine whether asubject with the mutation is at risk for a disorder caused by themutation. Mutations include deletion, addition, or substitution of oneor more nucleotides in the gene, chromosomal rearrangement, such asinversion or transposition, modification of genomic DNA, such asaberrant methylation patterns or changes in gene copy number, such asamplification. Detection of a mutated form of the protease geneassociated with a dysfunction provides a diagnostic tool for an activedisease or susceptibility to disease when the disease results fromoverexpression, underexpression, or altered expression of a proteaseprotein.

[0144] Individuals carrying mutations in the protease gene can bedetected at the nucleic acid level by a variety of techniques. GenomicDNA can be analyzed directly or can be amplified by using PCR prior toanalysis. RNA or cDNA can be used in the same way. In some uses,detection of the mutation involves the use of a probe/primer in apolymerase chain reaction (PCR) (see, e.g. U.S. Pat. Nos. 4,683,195 and4,683,202), such as anchor PCR or RACE PCR, or, alternatively, in aligation chain reaction (LCR) (see, e.g., Landegran et al., Science241:1077-1080 (1988); and Nakazawa et al., PNAS 91:360-364 (1994)), thelatter of which can be particularly useful for detecting point mutationsin the gene (see Abravaya et al., Nucleic Acids Res. 23:675-682 (1995)).This method can include the steps of collecting a sample of cells from apatient, isolating nucleic acid (e.g., genomic, mRNA or both) from thecells of the sample, contacting the nucleic acid sample with one or moreprimers which specifically hybridize to a gene under conditions suchthat hybridization and amplification of the gene (if present) occurs,and detecting the presence or absence of an amplification product, ordetecting the size of the amplification product and comparing the lengthto a control sample. Deletions and insertions can be detected by achange in size of the amplified product compared to the normal genotype.Point mutations can be identified by hybridizing amplified DNA to normalRNA or antisense DNA sequences.

[0145] Alternatively, mutations in a protease gene can be directlyidentified, for example, by alterations in restriction enzyme digestionpatterns determined by gel electrophoresis.

[0146] Further, sequence-specific ribozymes (U.S. Pat. No. 5,498,531)can be used to score for the presence of specific mutations bydevelopment or loss of a ribozyme cleavage site. Perfectly matchedsequences can be distinguished from mismatched sequences by nucleasecleavage digestion assays or by differences in melting temperature.

[0147] Sequence changes at specific locations can also be assessed bynuclease protection assays such as RNase and S1 protection or thechemical cleavage method. Furthermore, sequence differences between amutant protease gene and a wild-type gene can be determined by directDNA sequencing. A variety of automated sequencing procedures can beutilized when performing the diagnostic assays (Naeve, C. W., (1995)Biotechniques 19:448), including sequencing by mass spectrometry (see,e.g., PCT International Publication No. WO 94/16101; Cohen et al., Adv.Chromatogr. 36:127-162 (1996); and Griffin et al., Appl. Biochem.Biotechnol. 38:147-159 (1993)).

[0148] Other methods for detecting mutations in the gene include methodsin which protection from cleavage agents is used to detect mismatchedbases in RNA/RNA or RNA/DNA duplexes (Myers et al., Science 230:1242(1985)); Cotton et al., PNAS 85:4397 (1988); Saleeba et al., Meth.Enzymol. 217:286-295 (1992)), electrophoretic mobility of mutant andwild type nucleic acid is compared (Orita et al., PNAS 86:2766 (1989);Cotton et al., Mutat. Res. 285:125-144 (1993); and Hayashi et al.,Genet. Anal. Tech. Appl. 9:73-79 (1992)), and movement of mutant orwild-type fragments in polyacrylamide gels containing a gradient ofdenaturant is assayed using denaturing gradient gel electrophoresis(Myers et al., Nature 313:495 (1985)). Examples of other techniques fordetecting point mutations include selective oligonucleotidehybridization, selective amplification, and selective primer extension.

[0149] The nucleic acid molecules are also useful for testing anindividual for a genotype that while not necessarily causing thedisease, nevertheless affects the treatment modality. Thus, the nucleicacid molecules can be used to study the relationship between anindividual's genotype and the individual's response to a compound usedfor treatment (pharmacogenomic relationship). Accordingly, the nucleicacid molecules described herein can be used to assess the mutationcontent of the protease gene in an individual in order to select anappropriate compound or dosage regimen for treatment.

[0150] Thus nucleic acid molecules displaying genetic variations thataffect treatment provide a diagnostic target that can be used to tailortreatment in an individual. Accordingly, the production of recombinantcells and animals containing these polymorphisms allow effectiveclinical design of treatment compounds and dosage regimens.

[0151] The nucleic acid molecules are thus useful as antisenseconstructs to control protease gene expression in cells, tissues, andorganisms. A DNA antisense nucleic acid molecule is designed to becomplementary to a region of the gene involved in transcription,preventing transcription and hence production of protease protein. Anantisense RNA or DNA nucleic acid molecule would hybridize to the mRNAand thus block translation of mRNA into protease protein. Alternatively,a class of antisense molecules can be used to inactivate mRNA in orderto decrease expression of protease nucleic acid. Accordingly, thesemolecules can treat a disorder characterized by abnormal or undesiredprotease nucleic acid expression. This technique involves cleavage bymeans of ribozymes containing nucleotide sequences complementary to oneor more regions in the mRNA that attenuate the ability of the mRNA to betranslated. Possible regions include coding regions and particularlycoding regions corresponding to the catalytic and other functionalactivities of the protease protein, such as substrate binding.

[0152] The nucleic acid molecules also provide vectors for gene therapyin patients containing cells that are aberrant in protease geneexpression. Thus, recombinant cells, which include the patient's cellsthat have been engineered ex vivo and returned to the patient, areintroduced into an individual where the cells produce the desiredprotease protein to treat the individual.

[0153] The invention also encompasses kits for detecting the presence ofa protease nucleic acid in a biological sample. For example, the kit cancomprise reagents such as a labeled or labelable nucleic acid or agentcapable of detecting protease nucleic acid in a biological sample; meansfor determining the amount of protease nucleic acid in the sample; andmeans for comparing the amount of protease nucleic acid in the samplewith a standard. The compound or agent can be packaged in a suitablecontainer. The kit can further comprise instructions for using the kitto detect protease protein mRNA or DNA.

[0154] Nucleic Acid Arrays

[0155] The present invention further provides nucleic acid detectionkits, such as arrays or microarrays of nucleic acid molecules that arebased on the sequence information provided in FIG. 1 (SEQ ID NO:1).

[0156] As used herein “arrays” or “microarrays” refers to an array ofdistinct polynucleotides or oligonucleotides synthesized on a substrate,such as paper, nylon or other type of membrane, filter, chip, glassslide, or any other suitable solid support. In one embodiment, themicroarray is prepared and used according to the methods described inU.S. Pat. No. 5,837,832, Chee et al., PCT application WO95/11995 (Cheeet al.), Lockhart, D. J. et al. (1996; Nat. Biotech. 14: 1675-1680) andSchena, M. et al. (1996; Proc. Natl. Acad. Sci. 93: 10614-10619), all ofwhich are incorporated herein in their entirety by reference. In otherembodiments, such arrays are produced by the methods described by Brownet al., U.S. Pat. No. 5,807,522.

[0157] The microarray or detection kit is preferably composed of a largenumber of unique, single-stranded nucleic acid sequences, usually eithersynthetic antisense oligonucleotides or fragments of cDNAs, fixed to asolid support. The oligonucleotides are preferably about 6-60nucleotides in length, more preferably 15-30 nucleotides in length, andmost preferably about 20-25 nucleotides in length. For a certain type ofmicroarray or detection kit, it may be preferable to useoligonucleotides that are only 7-20 nucleotides in length. Themicroarray or detection kit may contain oligonucleotides that cover theknown 5′, or 3′, sequence, sequential oligonucleotides which cover thefull length sequence; or unique oligonucleotides selected fromparticular areas along the length of the sequence. Polynucleotides usedin the microarray or detection kit may be oligonucleotides that arespecific to a gene or genes of interest.

[0158] In order to produce oligonucleotides to a known sequence for amicroarray or detection kit, the gene(s) of interest (or an ORFidentified from the contigs of the present invention) is typicallyexamined using a computer algorithm which starts at the 5′ or at the 3′end of the nucleotide sequence. Typical algorithms will then identifyoligomers of defined length that are unique to the gene, have a GCcontent within a range suitable for hybridization, and lack predictedsecondary structure that may interfere with hybridization. In certainsituations it may be appropriate to use pairs of oligonucleotides on amicroarray or detection kit. The “pairs” will be identical, except forone nucleotide that preferably is located in the center of the sequence.The second oligonucleotide in the pair (mismatched by one) serves as acontrol. The number of oligonucleotide pairs may range from two to onemillion. The oligomers are synthesized at designated areas on asubstrate using a light-directed chemical process. The substrate may bepaper, nylon or other type of membrane, filter, chip, glass slide or anyother suitable solid support.

[0159] In another aspect, an oligonucleotide may be synthesized on thesurface of the substrate by using a chemical coupling procedure and anink jet application apparatus, as described in PCT applicationWO95/251116 (Baldeschweiler et al.) which is incorporated herein in itsentirety by reference. In another aspect, a “gridded” array analogous toa dot (or slot) blot may be used to arrange and link cDNA fragments oroligonucleotides to the surface of a substrate using a vacuum system,thermal, UV, mechanical or chemical bonding procedures. An array, suchas those described above, may be produced by hand or by using availabledevices (slot blot or dot blot apparatus), materials (any suitable solidsupport), and machines (including robotic instruments), and may contain8, 24, 96, 384, 1536, 6144 or more oligonucleotides, or any other numberbetween two and one million which lends itself to the efficient use ofcommercially available instrumentation.

[0160] In order to conduct sample analysis using a microarray ordetection kit, the RNA or DNA from a biological sample is made intohybridization probes. The mRNA is isolated, and cDNA is produced andused as a template to make antisense RNA (aRNA). The aRNA is amplifiedin the presence of fluorescent nucleotides, and labeled probes areincubated with the microarray or detection kit so that the probesequences hybridize to complementary oligonucleotides of the microarrayor detection kit. Incubation conditions are adjusted so thathybridization occurs with precise complementary matches or with variousdegrees of less complementarity. After removal of nonhybridized probes,a scanner is used to determine the levels and patterns of fluorescence.The scanned images are examined to determine degree of complementarityand the relative abundance of each oligonucleotide sequence on themicroarray or detection kit. The biological samples may be obtained fromany bodily fluids (such as blood, urine, saliva, phlegm, gastric juices,etc.), cultured cells, biopsies, or other tissue preparations. Adetection system may be used to measure the absence, presence, andamount of hybridization for all of the distinct sequencessimultaneously. This data may be used for large-scale correlationstudies on the sequences, expression patterns, mutations, variants, orpolymorphisms among samples.

[0161] Using such arrays, the present invention provides methods toidentify the expression of the protease proteins/peptides of the presentinvention. In detail, such methods comprise incubating a test samplewith one or more nucleic acid molecules and assaying for binding of thenucleic acid molecule with components within the test sample. Suchassays will typically involve arrays comprising many genes, at least oneof which is a gene of the present invention and or alleles of theprotease gene of the present invention.

[0162] Conditions for incubating a nucleic acid molecule with a testsample vary. Incubation conditions depend on the format employed in theassay, the detection methods employed, and the type and nature of thenucleic acid molecule used in the assay. One skilled in the art willrecognize that any one of the commonly available hybridization,amplification or array assay formats can readily be adapted to employthe novel fragments of the Human genome disclosed herein. Examples ofsuch assays can be found in Chard, T, An Introduction toRadioimmunoassay and Related Techniques, Elsevier Science Publishers,Amsterdam, The Netherlands (1986); Bullock, G. R. et al., Techniques inImmunocytochemistry, Academic Press, Orlando, Fla. Vol. 1 (1982), Vol. 2(1983), Vol. 3 (1985); Tijssen, P., Practice and Theory of EnzymeImmunoassays: Laboratory Techniques in Biochemistry and MolecularBiology, Elsevier Science Publishers, Amsterdam, The Netherlands (1985).

[0163] The test samples of the present invention include cells, proteinor membrane extracts of cells. The test sample used in theabove-described method will vary based on the assay format, nature ofthe detection method and the tissues, cells or extracts used as thesample to be assayed. Methods for preparing nucleic acid extracts or ofcells are well known in the art and can be readily be adapted in orderto obtain a sample that is compatible with the system utilized.

[0164] In another embodiment of the present invention, kits are providedwhich contain the necessary reagents to carry out the assays of thepresent invention.

[0165] Specifically, the invention provides a compartmentalized kit toreceive, in close confinement, one or more containers which comprises:(a) a first container comprising one of the nucleic acid molecules thatcan bind to a fragment of the Human genome disclosed herein; and (b) oneor more other containers comprising one or more of the following: washreagents, reagents capable of detecting presence of a bound nucleicacid.

[0166] In detail, a compartmentalized kit includes any kit in whichreagents are contained in separate containers. Such containers includesmall glass containers, plastic containers, strips of plastic, glass orpaper, or arraying material such as silica. Such containers allows oneto efficiently transfer reagents from one compartment to anothercompartment such that the samples and reagents are notcross-contaminated, and the agents or solutions of each container can beadded in a quantitative fashion from one compartment to another. Suchcontainers will include a container which will accept the test sample, acontainer which contains the nucleic acid probe, containers whichcontain wash reagents (such as phosphate buffered saline, Tris-buffers,etc.), and containers which contain the reagents used to detect thebound probe. One skilled in the art will readily recognize that thepreviously unidentified protease gene of the present invention can beroutinely identified using the sequence information disclosed herein canbe readily incorporated into one of the established kit formats whichare well known in the art, particularly expression arrays.

[0167] Vectors/Host Cells

[0168] The invention also provides vectors containing the nucleic acidmolecules described herein. The term “vector” refers to a vehicle,preferably a nucleic acid molecule, which can transport the nucleic acidmolecules. When the vector is a nucleic acid molecule, the nucleic acidmolecules are covalently linked to the vector nucleic acid. With thisaspect of the invention, the vector includes a plasmid, single or doublestranded phage, a single or double stranded RNA or DNA viral vector, orartificial chromosome, such as a BAC, PAC, YAC, OR MAC.

[0169] A vector can be maintained in the host cell as anextrachromosomal element where it replicates and produces additionalcopies of the nucleic acid molecules. Alternatively, the vector mayintegrate into the host cell genome and produce additional copies of thenucleic acid molecules when the host cell replicates.

[0170] The invention provides vectors for the maintenance (cloningvectors) or vectors for expression (expression vectors) of the nucleicacid molecules. The vectors can function in prokaryotic or eukaryoticcells or in both (shuttle vectors).

[0171] Expression vectors contain cis-acting regulatory regions that areoperably linked in the vector to the nucleic acid molecules such thattranscription of the nucleic acid molecules is allowed in a host cell.The nucleic acid molecules can be introduced into the host cell with aseparate nucleic acid molecule capable of affecting transcription. Thus,the second nucleic acid molecule may provide a trans-acting factorinteracting with the cis-regulatory control region to allowtranscription of the nucleic acid molecules from the vector.Alternatively, a trans-acting factor may be supplied by the host cell.Finally, a trans-acting factor can be produced from the vector itself.It is understood, however, that in some embodiments, transcriptionand/or translation of the nucleic acid molecules can occur in acell-free system.

[0172] The regulatory sequence to which the nucleic acid moleculesdescribed herein can be operably linked include promoters for directingmRNA transcription. These include, but are not limited to, the leftpromoter from bacteriophage λ, the lac, TRP, and TAC promoters from E.Coli, the early and late promoters from SV40, the CMV immediate earlypromoter, the adenovirus early and late promoters, and retroviruslong-terminal repeats.

[0173] In addition to control regions that promote transcription,expression vectors may also include regions that modulate transcription,such as repressor binding sites and enhancers. Examples include the SV40enhancer, the cytomegalovirus immediate early enhancer, polyomaenhancer, adenovirus enhancers, and retrovirus LTR enhancers.

[0174] In addition to containing sites for transcription initiation andcontrol, expression vectors can also contain sequences necessary fortranscription termination and, in the transcribed region a ribosomebinding site for translation. Other regulatory control elements forexpression include initiation and termination codons as well aspolyadenylation signals. The person of ordinary skill in the art wouldbe aware of the numerous regulatory sequences that are useful inexpression vectors. Such regulatory sequences are described, forexample, in Sambrook et al., Molecular Cloning: A Laboratory Manual.2nd. ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,(1989).

[0175] A variety of expression vectors can be used to express a nucleicacid molecule. Such vectors include chromosomal, episomal, andvirus-derived vectors, for example vectors derived from bacterialplasmids, from bacteriophage, from yeast episomes, from yeastchromosomal elements, including yeast artificial chromosomes, fromviruses such as baculoviruses, papovaviruses such as SV40, Vacciniaviruses, adenoviruses, poxviruses, pseudorabies viruses, andretroviruses. Vectors may also be derived from combinations of thesesources such as those derived from plasmid and bacteriophage geneticelements, e.g. cosmids and phagemids. Appropriate cloning and expressionvectors for prokaryotic and eukaryotic hosts are described in Sambrooket al., Molecular Cloning: A Laboratory Manual. 2nd. ed., Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., (1989).

[0176] The regulatory sequence may provide constitutive expression inone or more host cells (i.e. tissue specific) or may provide forinducible expression in one or more cell types such as by temperature,nutrient additive, or exogenous factor such as a hormone or otherligand. A variety of vectors providing for constitutive and inducibleexpression in prokaryotic and eukaryotic hosts are well known to thoseof ordinary skill in the art.

[0177] The nucleic acid molecules can be inserted into the vectornucleic acid by well-known methodology. Generally, the DNA sequence thatwill ultimately be expressed is joined to an expression vector bycleaving the DNA sequence and the expression vector with one or morerestriction enzymes and then ligating the fragments together. Proceduresfor restriction enzyme digestion and ligation are well known to those ofordinary skill in the art.

[0178] The vector containing the appropriate nucleic acid molecule canbe introduced into an appropriate host cell for propagation orexpression using well-known techniques. Bacterial cells include, but arenot limited to, E. coli, Streptomyces, and Salmonella typhimurium.Eukaryotic cells include, but are not limited to, yeast, insect cellssuch as Drosophila, animal cells such as COS and CHO cells, and plantcells.

[0179] As described herein, it may be desirable to express the peptideas a fusion protein. Accordingly, the invention provides fusion vectorsthat allow for the production of the peptides. Fusion vectors canincrease the expression of a recombinant protein, increase thesolubility of the recombinant protein, and aid in the purification ofthe protein by acting for example as a ligand for affinity purification.A proteolytic cleavage site may be introduced at the junction of thefusion moiety so that the desired peptide can ultimately be separatedfrom the fusion moiety. Proteolytic enzymes include, but are not limitedto, factor Xa, thrombin, and enteroprotease. Typical fusion expressionvectors include pGEX (Smith et al., Gene 67:31-40 (1988)), pMAL (NewEngland Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.)which fuse glutathione S-transferase (GST), maltose E binding protein,or protein A, respectively, to the target recombinant protein. Examplesof suitable inducible non-fusion E. coli expression vectors include pTrc(Amann et al., Gene 69:301-315 (1988)) and pET 11d (Studier et al., GeneExpression Technology: Methods in Enzymology 185:60-89 (1990)).

[0180] Recombinant protein expression can be maximized in host bacteriaby providing a genetic background wherein the host cell has an impairedcapacity to proteolytically cleave the recombinant protein. (Gottesman,S., Gene Expression Technology: Methods in Enzymology 185, AcademicPress, San Diego, Calif. (1990)119-128). Alternatively, the sequence ofthe nucleic acid molecule of interest can be altered to providepreferential codon usage for a specific host cell, for example E. coli.(Wada et al., Nucleic Acids Res. 20:2111-2118 (1992)).

[0181] The nucleic acid molecules can also be expressed by expressionvectors that are operative in yeast. Examples of vectors for expressionin yeast e.g., S. cerevisiae include pYepSec1 (Baldari, et al., EMBO J.6:229-234 (1987)), pMFa (Kujan et al., Cell 30:933-943(1982)), pJRY88(Schultz et al., Gene 54:113-123 (1987)), and pYES2 (InvitrogenCorporation, San Diego, Calif.).

[0182] The nucleic acid molecules can also be expressed in insect cellsusing, for example, baculovirus expression vectors. Baculovirus vectorsavailable for expression of proteins in cultured insect cells (e.g., Sf9cells) include the pAc series (Smith et al., Mol. Cell Biol. 3:2156-2165(1983)) and the pVL series (Lucklow et al., Virology 170:31-39 (1989)).

[0183] In certain embodiments of the invention, the nucleic acidmolecules described herein are expressed in mammalian cells usingmammalian expression vectors. Examples of mammalian expression vectorsinclude pCDM8 (Seed, B. Nature 329:840(1987)) and pMT2PC (Kaufman etal., EMBO J. 6:187-195 (1987)).

[0184] The expression vectors listed herein are provided by way ofexample only of the well-known vectors available to those of ordinaryskill in the art that would be useful to express the nucleic acidmolecules. The person of ordinary skill in the art would be aware ofother vectors suitable for maintenance propagation or expression of thenucleic acid molecules described herein. These are found for example inSambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: ALaboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.

[0185] The invention also encompasses vectors in which the nucleic acidsequences described herein are cloned into the vector in reverseorientation, but operably linked to a regulatory sequence that permitstranscription of antisense RNA. Thus, an antisense transcript can beproduced to all, or to a portion, of the nucleic acid molecule sequencesdescribed herein, including both coding and non-coding regions.Expression of this antisense RNA is subject to each of the parametersdescribed above in relation to expression of the sense RNA (regulatorysequences, constitutive or inducible expression, tissue-specificexpression).

[0186] The invention also relates to recombinant host cells containingthe vectors described herein. Host cells therefore include prokaryoticcells, lower eukaryotic cells such as yeast, other eukaryotic cells suchas insect cells, and higher eukaryotic cells such as mammalian cells.

[0187] The recombinant host cells are prepared by introducing the vectorconstructs described herein into the cells by techniques readilyavailable to the person of ordinary skill in the art. These include, butare not limited to, calcium phosphate transfection,DEAE-dextran-mediated transfection, cationic lipid-mediatedtransfection, electroporation, transduction, infection, lipofection, andother techniques such as those found in Sambrook, et al. (MolecularCloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).

[0188] Host cells can contain more than one vector. Thus, differentnucleotide sequences can be introduced on different vectors of the samecell. Similarly, the nucleic acid molecules can be introduced eitheralone or with other nucleic acid molecules that are not related to thenucleic acid molecules such as those providing trans-acting factors forexpression vectors. When more than one vector is introduced into a cell,the vectors can be introduced independently, co-introduced or joined tothe nucleic acid molecule vector.

[0189] In the case of bacteriophage and viral vectors, these can beintroduced into cells as packaged or encapsulated virus by standardprocedures for infection and transduction. Viral vectors can bereplication-competent or replication-defective. In the case in whichviral replication is defective, replication will occur in host cellsproviding functions that complement the defects.

[0190] Vectors generally include selectable markers that enable theselection of the subpopulation of cells that contain the recombinantvector constructs. The marker can be contained in the same vector thatcontains the nucleic acid molecules described herein or may be on aseparate vector. Markers include tetracycline or ampicillin-resistancegenes for prokaryotic host cells and dihydrofolate reductase or neomycinresistance for eukaryotic host cells. However, any marker that providesselection for a phenotypic trait will be effective.

[0191] While the mature proteins can be produced in bacteria, yeast,mammalian cells, and other cells under the control of the appropriateregulatory sequences, cell-free transcription and translation systemscan also be used to produce these proteins using RNA derived from theDNA constructs described herein.

[0192] Where secretion of the peptide is desired, which is difficult toachieve with multi-transmembrane domain containing proteins such asproteases, appropriate secretion signals are incorporated into thevector. The signal sequence can be endogenous to the peptides orheterologous to these peptides.

[0193] Where the peptide is not secreted into the medium, which istypically the case with proteases, the protein can be isolated from thehost cell by standard disruption procedures, including freeze thaw,sonication, mechanical disruption, use of lysing agents and the like.The peptide can then be recovered and purified by well-knownpurification methods including ammonium sulfate precipitation, acidextraction, anion or cationic exchange chromatography, phosphocellulosechromatography, hydrophobic-interaction chromatography, affinitychromatography, hydroxylapatite chromatography, lectin chromatography,or high performance liquid chromatography.

[0194] It is also understood that depending upon the host cell inrecombinant production of the peptides described herein, the peptidescan have various glycosylation patterns, depending upon the cell, ormaybe non-glycosylated as when produced in bacteria. In addition, thepeptides may include an initial modified methionine in some cases as aresult of a host-mediated process.

[0195] Uses of Vectors and Host Cells

[0196] The recombinant host cells expressing the peptides describedherein have a variety of uses. First, the cells are useful for producinga protease protein or peptide that can be further purified to producedesired amounts of protease protein or fragments. Thus, host cellscontaining expression vectors are useful for peptide production.

[0197] Host cells are also useful for conducting cell-based assaysinvolving the protease protein or protease protein fragments, such asthose described above as well as other formats known in the art. Thus, arecombinant host cell expressing a native protease protein is useful forassaying compounds that stimulate or inhibit protease protein function.

[0198] Host cells are also useful for identifying protease proteinmutants in which these functions are affected. If the mutants naturallyoccur and give rise to a pathology, host cells containing the mutationsare useful to assay compounds that have a desired effect on the mutantprotease protein (for example, stimulating or inhibiting function) whichmay not be indicated by their effect on the native protease protein.

[0199] Genetically engineered host cells can be further used to producenon-human transgenic animals. A transgenic animal is preferably amammal, for example a rodent, such as a rat or mouse, in which one ormore of the cells of the animal include a transgene. A transgene isexogenous DNA, which is integrated into the genome of a cell from whicha transgenic animal develops and which remains in the genome of themature animal in one or more cell types or tissues of the transgenicanimal. These animals are useful for studying the function of a proteaseprotein and identifying and evaluating modulators of protease proteinactivity. Other examples of transgenic animals include non-humanprimates, sheep, dogs, cows, goats, chickens, and amphibians.

[0200] A transgenic animal can be produced by introducing nucleic acidinto the male pronuclei of a fertilized oocyte, e.g., by microinjection,retroviral infection, and allowing the oocyte to develop in apseudopregnant female foster animal. Any of the protease proteinnucleotide sequences can be introduced as a transgene into the genome ofa non-human animal, such as a mouse.

[0201] Any of the regulatory or other sequences useful in expressionvectors can form part of the transgenic sequence. This includes intronicsequences and polyadenylation signals, if not already included. Atissue-specific regulatory sequence(s) can be operably linked to thetransgene to direct expression of the protease protein to particularcells.

[0202] Methods for generating transgenic animals via embryo manipulationand microinjection, particularly animals such as mice, have becomeconventional in the art and are described, for example, in U.S. Pat.Nos. 4,736,866 and 4,870,009, both by Leder et al., U.S. Pat. No.4,873,191 by Wagner et al. and in Hogan, B., Manipulating the MouseEmbryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1986). Similar methods are used for production of other transgenicanimals. A transgenic founder animal can be identified based upon thepresence of the transgene in its genome and/or expression of transgenicmRNA in tissues or cells of the animals. A transgenic founder animal canthen be used to breed additional animals carrying the transgene.Moreover, transgenic animals carrying a transgene can further be bred toother transgenic animals carrying other transgenes. A transgenic animalalso includes animals in which the entire animal or tissues in theanimal have been produced using the homologously recombinant host cellsdescribed herein.

[0203] In another embodiment, transgenic non-human animals can beproduced which contain selected systems that allow for regulatedexpression of the transgene. One example of such a system is thecre/loxP recombinase system of bacteriophage P1. For a description ofthe cre/loxP recombinase system, see, e.g., Lakso et al. PNAS89:6232-6236 (1992). Another example of a recombinase system is the FLPrecombinase system of S. cerevisiae (O'Gorman et al. Science251:1351-1355 (1991). If a cre/loxP recombinase system is used toregulate expression of the transgene, animals containing transgenesencoding both the Cre recombinase and a selected protein is required.Such animals can be provided through the construction of “double”transgenic animals, e.g., by mating two transgenic animals, onecontaining a transgene encoding a selected protein and the othercontaining a transgene encoding a recombinase.

[0204] Clones of the non-human transgenic animals described herein canalso be produced according to the methods described in Wilmut, I. et al.Nature 385:810-813 (1997) and PCT International Publication Nos. WO97/07668 and WO 97/07669. In brief, a cell, e.g., a somatic cell, fromthe transgenic animal can be isolated and induced to exit the growthcycle and enter G_(o) phase. The quiescent cell can then be fused, e.g.,through the use of electrical pulses, to an enucleated oocyte from ananimal of the same species from which the quiescent cell is isolated.The reconstructed oocyte is then cultured such that it develops tomorula or blastocyst and then transferred to pseudopregnant femalefoster animal. The offspring born of this female foster animal will be aclone of the animal from which the cell, e.g., the somatic cell, isisolated.

[0205] Transgenic animals containing recombinant cells that express thepeptides described herein are useful to conduct the assays describedherein in an in vivo context. Accordingly, the various physiologicalfactors that are present in vivo and that could effect substratebinding, protease protein activity/activation, and signal transduction,may not be evident from in vitro cell-free or cell-based assays.Accordingly, it is useful to provide non-human transgenic animals toassay in vivo protease protein function, including substrateinteraction, the effect of specific mutant protease proteins on proteaseprotein function and substrate interaction, and the effect of chimericprotease proteins. It is also possible to assess the effect of nullmutations, that is mutations that substantially or completely eliminateone or more protease protein functions.

EXAMPLES

[0206] Materials

[0207] Taq DNA polymerase was obtained from Perkin-Elmer (Norwalk,Conn.). T4 DNA ligase and restriction enzymes were purchased from NewEngland Biolabs (Beverly, Mass.). Oligonucletide primers weresynthesized using an Applied Biosystems 394 DNA/RNA synthesizer or madeby Operon Technologies (Alameda, Calif.). The hepsin polyclonal antibodywas raised against a synthetic peptide that corresponds to the last 20amino acids of Hepsin. Spectrozyme PCa was purchased from AmericanDiagnostica (Greenwich, Conn.), and Glu-Pro-Arg-pNA was obtained fromChromogenix (Molndal, Sweeden).

[0208] Cloning of Human Hepsin

[0209] Human hepsin was PCR amplified from a human liver cDNA population(Clontech) with 5′hHepsin (5′CAGCCATGGCGCAGAAGGAGGGT) and 3′hHepsin(5′TGCTCTAGAGCTGGGTCACCATGCCGCT) primers. PCR amplification wasperformed using Taq DNA polymerase under standard conditions.Amplification was carried out for 30 cycles consisting of 1 min at 94°C., 1 min at 50° C. and 1.5 min at 72° C. The PCR product was thencloned into the pCR2.1 TOPO vector following the manufacturer'srecommendations. The hepsin sequence in pCR2.1TOPOhepsin was identicalto the published hepsin sequence (Leytus, S. P., et al, 1988).

[0210] Construction and Transformation of Hepsin Expression Vector

[0211] Recombinant hepsin, Hepsin 46, starts at Serine 46 right afterthe predicted transmembrane region (FIG. 1). pPIC9 Hepsin46 was made byPCR amplification of pCR2.1TOPOhepsin with 5′hHEP46Xho(5′CCCTCGAGAAAAGAAGTGACCAGGAGCCGCTGT) and 3′hHEPEco(CGGAATTCTAGAGCTGGGTCACCATGCCGCT) primers and cloning the product intothe XhoI and EcoRI site of pPIC9 (Invitrogen, FIG. 2). The hepsinsequence is cloned down-stream from, and in frame with, the α-factorsecretion signal of the vector allowing for the secreted expression ofhepsin. Hepsin 46 constructs containing a his tag at the amino (pPIC9Hep46N) end were made by amplifying pPIC9 Hepsin 46 with 5′hHEPhisXho(CTCGAGAAAAGACATCATCATCATCATCACAGTGACCAGGAGCCGCTGT).

[0212] Expression constructs were linearized with SalI and transformedinto KM71 by electroporation. Clones were evaluated for the expressionof recombinant protein by Western blot analysis and cleavage ofchromogenic substrates.

[0213] Expression and Purification of His-Hepsin46 in Pichia pastoris

[0214] For small-scale purification, the hepsin clone was grown in 4LBMGY for 48 hrs at 30° C. and induced in 2 L BMMY containing 1% Methanol@ OD₆₀₀˜80 OD/ml for 72 hrs. In a large-scale purification, a 6L cultureof the hepsin clone was grown overnight in BMGY and induced with aglycerol/methanol (37.5%/25%) mixed feed for 72 hrs. Hepsin46 appears tobe expressed in both the zymogen and active form in the culturesupernatant. However, during purification, hepsin is completelyconverted to the active, disulfide linked two-chain form, consisting ofa 22 kDa light chain and a 32 kDa heavy chain. The culture supernatantwas filtered and buffer exchanged in 50 mM Sodium Acetate, pH 5.5. Thebuffer-exchanged starting material was loaded on a 24 ml SP sepharosecolumn (Pharmacia). The column was then washed with buffer A (50 mMSodium Acetate, pH 5.5), and the bound protein was eluted with a 1M NaClgradient in buffer A (FIG. 3). Fractions containing active protein werepooled and dialyzed in 10 mM Tris (pH 8.0), 0.3 M NaCl and 5 mMimidazole. The dialyzed sample was batch bound to 4 ml Ni-NTA Superflow(Qiagen) @ 4° C. for 1 hr, loaded into a column reservoir and washedwith dialysis buffer. The bound protein was step eluted with 50, 100 or250 mM imidazole in Tris (pH 8.0) buffer containing 0.3 M NaCl (FIG. 4).Recombinant hepsin eluted in the 50 and 100 mM Imidazole fractions. Thefractions were pooled and dialyzed in storage buffer (10 mM Tris, 0.1MNaCl, 10% glycerol, pH 8.0). The untagged recombinant hepsin waspurified from a small-scale culture using only the SP sepharose column.The fractions containing protein were pooled together and dialyzed instorage buffer as above. Recombinant hepsin purified with the abovemethod was 85% pure and had an overall yield of 0.3 mg/L (FIG. 5).

[0215] Enzyme Activity Assay

[0216] Enzyme activity was measured by monitoring the hydrolysis ofsynthetic substrates, Spectrozyme PCa (H-D-Lys-Pro-Arg-pNA) andGlu-Pro-Arg-pNA. Assays were performed at 22° C. in 100-ul reactionscontaining Tris-imidazole buffer (30 mM Tris, 30 mM Imidazole, 200 mMNaCl, pH 8.4) in 96 well, round-bottom microtiter plates (Falcon). Thehydrolysis of substrate (0.5 mM) was measured at 405 nm over 5 min usinga UV/MAX kinetic microplate reader (Molecular Devices, Sunnyvale,Calif.). The result shows while Hep 46 has enzymatic activity, whereasHep 143 and Hep 163 shows inactive enzymatic activity.

[0217] All publications and patents mentioned in the above specificationare herein incorporated by reference. Various modifications andvariations of the described method and system of the invention will beapparent to those skilled in the art without departing from the scopeand spirit of the invention. Although the invention has been describedin connection with specific preferred embodiments, it should beunderstood that the invention as claimed should not be unduly limited tosuch specific embodiments. Indeed, various modifications of theabove-described modes for carrying out the invention, which are obviousto those skilled in the field of molecular biology or related fields areintended to be within the scope of the following claims.

1 2 1 1254 DNA Homo sapiens 1 atggcgcaga aggagggtgg ccggactgtgccatgctgct ccagacccaa ggtggcagct 60 ctcactgcgg ggaccctgct acttctgacagccatcgggg cggcatcctg ggccattgtg 120 gctgttctcc tcaggagtga ccaggagccgctgtacccag tgcaggtcag ctctgcggac 180 gctcggctca tggtctttga caagacggaagggacgtggc ggctgctgtg ctcctcgcgc 240 tccaacgcca gggtagccgg actcagctgcgaggagatgg gcttcctcag ggcactgacc 300 cactccgagc tggacgtgcg aacggcgggcgccaatggca cgtcgggctt cttctgtgtg 360 gacgagggga ggctgcccca cacccagaggctgctggagg tcatctccgt gtgtgattgc 420 cccagaggcc gtttcttggc cgccatctgccaagactgtg gccgcaggaa gctgcccgtg 480 gaccgcatcg tgggaggccg ggacaccagcttgggccggt ggccgtggca agtcagcctt 540 cgctatgatg gagcacacct ctgtgggggatccctgctct ccggggactg ggtgctgaca 600 gccgcccact gcttcccgga gcggaaccgggtcctgtccc gatggcgagt gtttgccggt 660 gccgtggccc aggcctctcc ccacggtctgcagctggggg tgcaggctgt ggtctaccac 720 gggggctatc ttccctttcg ggaccccaacagcgaggaga acagcaacga tattgccctg 780 gtccacctct ccagtcccct gcccctcacagaatacatcc agcctgtgtg cctcccagct 840 gccggccagg ccctggtgga tggcaagatctgtaccgtga cgggctgggg caacacgcag 900 tactatggcc aacaggccgg ggtactccaggaggctcgag tccccataat cagcaatgat 960 gtctgcaatg gcgctgactt ctatggaaaccagatcaagc ccaagatgtt ctgtgctggc 1020 taccccgagg gtggcattga tgcctgccagggcgacagcg gtggtccctt tgtgtgtgag 1080 gacagcatct ctcggacgcc acgttggcggctgtgtggca ttgtgagttg gggcactggc 1140 tgtgccctgg cccagaagcc aggcgtctacaccaaagtca gtgacttccg ggagtggatc 1200 ttccaggcca taaagactca ctccgaagccagcggcatgg tgacccagct ctga 1254 2 380 PRT Homo sapiens 2 His His His HisHis His His His Ser Asp Gln Glu Pro Leu Tyr Pro 1 5 10 15 Val Gln ValSer Ser Ala Asp Ala Arg Leu Met Val Phe Asp Lys Thr 20 25 30 Glu Gly ThrTrp Arg Leu Leu Cys Ser Ser Arg Ser Asn Ala Arg Val 35 40 45 Ala Gly LeuSer Cys Glu Glu Met Gly Phe Leu Arg Ala Leu Thr His 50 55 60 Ser Glu LeuAsp Val Arg Thr Ala Gly Ala Asn Gly Thr Ser Gly Phe 65 70 75 80 Phe CysVal Asp Glu Gly Arg Leu Pro His Thr Gln Arg Leu Leu Glu 85 90 95 Val IleSer Val Cys Asp Cys Pro Arg Gly Arg Phe Leu Ala Ala Ile 100 105 110 CysGln Asp Cys Gly Arg Arg Lys Leu Pro Val Asp Arg Ile Val Gly 115 120 125Gly Arg Asp Thr Ser Leu Gly Arg Trp Pro Trp Gln Val Ser Leu Arg 130 135140 Tyr Asp Gly Ala His Leu Cys Gly Gly Ser Leu Leu Ser Gly Asp Trp 145150 155 160 Val Leu Thr Ala Ala His Cys Phe Pro Glu Arg Asn Arg Val LeuSer 165 170 175 Arg Trp Arg Val Phe Ala Gly Ala Val Ala Gln Ala Ser ProHis Gly 180 185 190 Leu Gln Leu Gly Val Gln Ala Val Val Tyr His Gly GlyTyr Leu Pro 195 200 205 Phe Arg Asp Pro Asn Ser Glu Glu Asn Ser Asn AspIle Ala Leu Val 210 215 220 His Leu Ser Ser Pro Leu Pro Leu Thr Glu TyrIle Gln Pro Val Cys 225 230 235 240 Leu Pro Ala Ala Gly Gln Ala Leu ValAsp Gly Lys Ile Cys Thr Val 245 250 255 Thr Gly Trp Gly Asn Thr Gln TyrTyr Gly Gln Gln Ala Gly Val Leu 260 265 270 Gln Glu Ala Arg Val Pro IleIle Ser Asn Asp Val Cys Asn Gly Ala 275 280 285 Asp Phe Tyr Gly Asn GlnIle Lys Pro Lys Met Phe Cys Ala Gly Tyr 290 295 300 Pro Glu Gly Gly IleAsp Ala Cys Gln Gly Asp Ser Gly Gly Pro Phe 305 310 315 320 Val Cys GluAsp Ser Ile Ser Arg Thr Pro Arg Trp Arg Leu Cys Gly 325 330 335 Ile ValSer Trp Gly Thr Gly Cys Ala Leu Ala Gln Lys Pro Gly Val 340 345 350 TyrThr Lys Val Ser Asp Phe Arg Glu Trp Ile Phe Gln Ala Ile Lys 355 360 365Thr His Ser Glu Ala Ser Gly Met Val Thr Gln Leu 370 375 380

That which is claimed is:
 1. An isolated peptide consisting of an aminoacid sequence selected from the group consisting of: (a) an amino acidsequence shown in SEQ ID NO:2; (b) an amino acid sequence shares atleast 95% percent homology with an amino acid sequence shown in SEQ IDNO:2; and (c) a fragment of an amino acid sequence shown in SEQ ID NO:2,wherein said fragment comprises at least 10 contiguous amino acids. 2.An isolated peptide comprising an amino acid sequence selected from thegroup consisting of: (a) an amino acid sequence shown in SEQ ID NO:2;(b) an amino acid sequence shares at least 95% percent homology with anamino acid sequence shown in SEQ ID NO:2; and (c) a fragment of an aminoacid sequence shown in SEQ ID NO:2, wherein said fragment comprises atleast 10 contiguous amino acids.
 3. An isolated antibody thatselectively binds to a peptide of claim
 2. 4. An isolated nucleic acidmolecule consisting of a nucleotide sequence selected from the groupconsisting of: (a) a nucleotide sequence that encodes an amino acidsequence shown in SEQ ID NO:2; (b) a nucleic acid sequence that sharesat least 90 percent homology with a nucleic acid sequence shown in SEQID NO:1; and (c) a nucleotide sequence that is the complement of anucleotide sequence of (a)-(b).
 5. An isolated nucleic acid moleculecomprising a nucleotide sequence selected from the group consisting of:(a) a nucleotide sequence that encodes an amino acid sequence shown inSEQ ID NO:2; (b) a nucleic acid sequence that shares at least 90 percenthomology with a nucleic acid sequence shown in SEQ ID NO:1; and (c) anucleotide sequence that is the complement of a nucleotide sequence of(a)-(b).
 6. A gene chip comprising a nucleic acid molecule of claim 5.7. A transgenic non-human animal comprising a nucleic acid molecule ofclaim
 5. 8. A nucleic acid vector comprising a nucleic acid molecule ofclaim
 5. 9. A host cell containing the vector of claim 8 which iscapable of production of SEQ ID NO:2.
 10. A method for producing any ofthe peptides of claim 1 comprising introducing a nucleotide sequenceencoding any of the amino acid sequences in (a)-(c) into a host cell,and culturing the host cell under conditions in which the peptides areexpressed from the nucleotide sequence.
 11. A method for producing anyof the peptides of claim 2 comprising introducing a nucleotide sequenceencoding any of the amino acid sequences in (a)-(c) into a host cell,and culturing the host cell under conditions in which the peptides areexpressed from the nucleotide sequence.
 12. A method for detecting thepresence of any of the peptides of claim 2 in a sample, said methodcomprising contacting said sample with a detection agent thatspecifically allows detection of the presence of the peptide in thesample and then detecting the presence of the peptide.
 13. A method fordetecting the presence of a nucleic acid molecule of claim 5 in asample, said method comprising contacting the sample with anoligonucleotide that hybridizes to said nucleic acid molecule understringent conditions and determining whether the oligonucleotide bindsto said nucleic acid molecule in the sample.
 14. A method foridentifying a modulator of a peptide of claim 2, said method comprisingcontacting said peptide with an agent and determining if said agent hasmodulated the function or activity of said peptide.
 15. The method ofclaim 14, wherein said agent is administered to a host cell comprisingan expression vector that expresses said peptide.
 16. A method foridentifying an agent that binds to any of the peptides of claim 2, saidmethod comprising contacting the peptide with an agent and assaying thecontacted mixture to determine whether a complex is formed with theagent bound to the peptide.
 17. A pharmaceutical composition comprisingan agent identified by the method of claim 16 and a pharmaceuticallyacceptable carrier therefor.
 18. A method for treating a disease orcondition mediated by a hepsin protease protein, said method comprisingadministering to a patient a pharmaceutically effective amount of anagent identified by the method of claim
 16. 19. A method for identifyinga modulator of the expression of a peptide of claim 2, said methodcomprising contacting a cell expressing said peptide with an agent, anddetermining if said agent has modulated the expression of said peptide.